JP6344766B2 - Method for producing cyan fluorescent protein with reduced pH sensitivity - Google Patents

Description

  The present invention proposes a method for producing a cyan fluorescent protein with reduced pH sensitivity.

  Fluorescent proteins (also called fluorescent probes), such as green fluorescent protein (green fluorescent protein or GFPav) and its variants extracted from Aequorea victoria jellyfish, are particularly fluorescent imaging techniques, flow cytometry And constitutes an indispensable tool for exploring live cells in high-throughput biological tests. When fused to various proteins, these fluorescent probes determine the localization and traffic movement of these proteins and perform various biological processes such as protein-protein interactions, enzyme activity or second messenger signaling. Allows analysis. The success of these proteins depends in particular on the fact that they can be expressed in numerous living and cellular compartments (eg nucleus, mitochondria, Golgi apparatus). Thus, a number of directed and random mutagenesis approaches performed in the last 15 years have generated a wide variety of variants of native GFPav (Day et al., 2009). These differ from GFPav through a few amino acid mutations and fluoresce in a different color range from blue to red.

  The most frequently used GFPav variants include enhanced cyan fluorescent protein (ECFPav or ECFP). This protein has 239 amino acids, 6-9 of which are mutated compared to native GFPav (K26R, S65T, Y66W, F64L, N146I, M153T, V163A, N164H, H231L) (Cubitt 1995; Llopis et al., 1998; Cubitt et al., 1999). Substitution of Tyr66 amino acid with tryptophan resulted in a shift of the excitation and emission spectra to blue at 436 and 475 nm, respectively, compared to GFPav, but other mutations caused protein folding, solubility, photostability and It was possible to enhance some physicochemical properties such as brightness. Nevertheless, the brightness of this ECFP remains below that of enhanced GFP (40%).

  To date, ECFP has been used in Forster-type resonance energy transfer (FRET) imaging studies, more specifically, as a result of partial overlap of their absorption and emission spectra, along with EYFP (Miyawaki et al., 1999) and its variants. , One of the most commonly used fluorescent proteins as a donor. Thus, the coupling of ECFP to this type of protein has made it possible to develop a large number of FRET biosensors, especially for cell metabolism studies.

  However, like all real-time imaging methods, FRET requires a very accurate quantitative analysis of the fluorescent signal, which is rare in the case of conventional microscopy techniques. Thus, the complex and less effective luminescent properties of ECFP do not allow reliable decoding of the results obtained by quantitative imaging in living cells. In fact, ECFP shows low quantum yield and short fluorescence lifetime compared to GFPav, in addition to some excited states that clearly indicate the existence of several distinct conformations that this protein can employ. Shaner et al., 2007; Patterson et al., 2001; Tramier et al., 2002).

For most fluorescent probes, ECFP is characterized by a very high sensitivity to environmental factors, especially pH (Miyawaki et al., 2000). Its average fluorescence lifetime (tau or τ) decreases slightly between pH 11 and pH 7, but significantly decreases from 2.5 ns to 1.5 ns when the pH drops to pH 5.5. It has also been well established that the fluorescence intensity (I _f ) decreases rapidly by about 10% at pH 6.5 and about 40% at pH 5.5. This pH sensitivity constitutes a major drawback in the use of ECFP in quantitative imaging in intact cells, as the intracellular pH varies depending on the cell compartment and experimental conditions to be tested, such as mitotic stimulation or metabolic stress. Thus, when ECFP is solubilized in two cell compartments at different pH (e.g. neutral pH in the cytosol and acidic pH in lysosomes), it has distinct luminescent properties independent of the biological process being studied. And potentially decipher artifacts. To date, the only way to reduce these artifacts is to periodically check the ambient pH or fix it ("clamping"). Therefore, it is highly desirable to have an ECFP that has reduced pH sensitivity and whose half-transition pH (pH _1/2 ) is well below that of physiological pH.

  Efforts made to date to improve the photophysical and physicochemical performance of ECFPs are to enhance its brightness (or fluorescence intensity), its maturation and solubilization, and its luminescent properties, especially its fluorescence emission. (Rizzo et al. (2004); Nguyen et al. (2005); Goedhart et al. (2010); Sawano et al. (2000)). Nonetheless, no particular research has focused on developing cyan fluorescent proteins that exhibit reduced pH sensitivity and preserve the fluorescence spectral properties of ECFP.

Sambrook et al., "Molecular Cloning: A laboratory Manual", 3rd edition, Cold Spring Harbor Laboratory Press, (2001) Ausubel et al., "Current Protocols in Molecular Biology", John Wiley & Sons (2011) http://www.kazusa.or.jp/codon/ B. Valeur, “Molecular Fluorescence: Principles and Applications”, 3rd edition. (2006), Wiley-VCH Needleman and Wunsch (J. Mol. Biol., 1970, 48: 443) American Type Culture Collection (ATCC) (www.ATCC.org) `` Baculovirus Expression vectors, A Laboratory Manual '', David R. O'Reilly et al., Oxford University Press, USA, (1992)

  Therefore, there is a need to develop methods that produce cyan fluorescent proteins with reduced pH sensitivity while preserving spectral characteristics that are specific for those proteins.

  The present invention proposes a method for producing cyan fluorescent protein with reduced pH sensitivity, in particular acidic pH. In addition to the cyan fluorescent protein obtained by the above method, another embodiment of the present invention includes a nucleic acid encoding the protein, a recombinant vector containing the nucleic acid, a host cell expressing the protein, and a bio containing the protein. It relates to the use of sensors and the proteins, nucleic acids, host cells and biosensors.

It is a figure which shows the pH dependence of the fluorescence characteristic of purified ECFPr (sequence number 4). Graph A: Absorption spectrum normalized to the same region. Graph B: Fluorescence emission spectrum normalized to the same region (λex = 420 nm). Graph C: Fluorescence intensity emitted at 474 nm (Δλ = 6 nm) normalized to the maximum value as a function of pH. Graph D: Fluorescence lifetime <τ> (ns) as a function of pH. It is a figure which shows the pH dependence of the fluorescence characteristic of purified ECFPr (148G). Graph A: Absorption spectrum normalized to the same region. Graph B: Fluorescence emission spectrum normalized to the same region (λex = 420 nm). Graph C: Fluorescence intensity emitted at 474 nm (Δλ = 6 nm) normalized to the maximum as a function of pH—comparison with ECFPr. Graph D: Comparison with fluorescence lifetime <τ> (ns) -ECFPr as a function of pH. It is a figure which shows the pH dependence of the fluorescence characteristic of purified ECFPr (148S). Graph A: Absorption spectrum normalized to the same region. Graph B: Fluorescence emission spectrum normalized to the same region (λex = 420 nm). Graph C: Fluorescence intensity emitted at 474 nm (Δλ = 6 nm) normalized to the maximum value—comparison with ECFPr. Graph D: Fluorescence lifetime vs. ECFPr. It is a figure which shows the pH dependence of the fluorescence characteristic of purified ECFPr (148A). Graph A: Absorption spectrum normalized to the same region. Graph B: Fluorescence emission spectrum normalized to the same region (λex = 420 nm). Graph C: Fluorescence intensity emitted at 474 nm (Δλ = 6 nm) normalized to the maximum value—comparison with ECFP. Graph D: Fluorescence lifetime vs. ECFPr. It is a figure which shows the pH dependence of the fluorescence characteristic of purified ECFPr (65S). Graph A: Absorption spectrum normalized to the same region. Graph B: Fluorescence emission spectrum normalized to the same region u (λex = 420 nm). Graph C: Fluorescence intensity emitted at 474 nm (Δλ = 6 nm) normalized to the maximum value—comparison with ECFPr. Graph D: Fluorescence lifetime vs. ECFPr. It is a figure which shows the pH dependence of the fluorescence characteristic of purified ECFPr (65S, 148G). Graph A: Absorption spectrum normalized to the same region. Graph B: Fluorescence emission spectrum normalized to the same region (λex = 420 nm). Graph C: Fluorescence intensity emitted at 474 nm (Δλ = 6 nm) normalized to the maximum value—comparison with ECFPr. Graph D: Fluorescence lifetime vs. ECFPr. It is a figure which shows the pH dependence of the fluorescence characteristic of purified ECFPr (72A, 145A, 148D). Graph A: Absorption spectrum normalized to the same region. Graph B: Fluorescence emission spectrum normalized to the same region (λex = 420 nm). Graph C: Fluorescence intensity emitted at 474 nm (Δλ = 6 nm) normalized to the maximum as a function of pH—comparison with ECFPr. Graph D: Comparison with fluorescence lifetime <τ> (ns) -ECFPr as a function of pH. It is a figure which shows the pH dependence of the fluorescence characteristic of purified ECFPr (72A, 145A, 148G). Graph A: Absorption spectrum normalized to the same region. Graph B: Fluorescence emission spectrum normalized to the same region (λex = 420 nm). Graph C: Fluorescence intensity emitted at 474 nm (Δλ = 6 nm) normalized to the maximum value—comparison with ECFPr. Graph D: Fluorescence lifetime vs. ECFPr. It is a figure which shows the pH dependence of the fluorescence characteristic of purified ECFPr (65S, 72A, 145A, 148D). Graph A: Absorption spectrum normalized to the same region. Graph B: Fluorescence emission spectrum normalized to the same region (λex = 420 nm). Graph C: Fluorescence intensity emitted at 474 nm (Δλ = 6 nm) normalized to the maximum value—comparison with ECFPr. Graph D: Fluorescence lifetime vs. ECFPr. It is a figure which shows the pH dependence of the fluorescence characteristic of purified ECFPr (65S, 72A, 148D, 206K). Graph A: Absorption spectrum normalized to the same region. Graph B: Fluorescence emission spectrum normalized to the same region (λex = 420 nm). Graph C: Fluorescence intensity emitted at 474 nm (Δλ = 6 nm) normalized to the maximum value—comparison with ECFPr. Graph D: Fluorescence lifetime vs. ECFPr. It is a figure which shows the pH dependence of the fluorescence characteristic of purified ECFPr (65S, 72A, 148G, 206K). Graph A: Absorption spectrum normalized to the same region. Graph B: Fluorescence emission spectrum normalized to the same region (λex = 420 nm). Graph C: Fluorescence intensity emitted at 474 nm (Δλ = 6 nm) normalized to the maximum value—comparison with ECFPr. Graph D: Fluorescence lifetime vs. ECFPr. It is a figure which shows the pH dependence of the fluorescence characteristic of purified ECFPr (65S, 72A, 206K). Graph A: Absorption spectrum normalized to the same region. Graph B: Fluorescence emission spectrum normalized to the same region (λex = 420 nm). Graph C: Fluorescence intensity emitted at 474 nm (Δλ = 6 nm) normalized to the maximum as a function of pH—comparison with ECFPr. Graph D: Comparison with fluorescence lifetime <τ> (ns) -ECFPr as a function of pH. It is a figure which shows the pH dependence of the fluorescence characteristic of purified ECFPr (148D). Graph A: Absorption spectrum normalized to the same region. Graph B: Fluorescence emission spectrum normalized to the same region (λex = 420 nm). Graph C: Fluorescence intensity emitted at 474 nm (Δλ = 6 nm) normalized to the maximum value—comparison with ECFPr. Graph D: Fluorescence lifetime vs. ECFPr. It is a figure which shows the pH dependence of the fluorescence characteristic of purified ECFPr (148N). Graph A: Absorption spectrum normalized to the same region. Graph B: Fluorescence emission spectrum normalized to the same region (λex = 420 nm). Graph C: Fluorescence intensity emitted at 474 nm (Δλ = 6 nm) normalized to the maximum value—comparison with ECFPr. It is a figure which shows the pH dependence of the fluorescence characteristic of purified ECFPr (148E). Graph A: Absorption spectrum normalized to the same region. Graph B: Fluorescence emission spectrum normalized to the same region (λex = 420 nm). Graph C: Fluorescence intensity emitted at 474 nm (Δλ = 6 nm) normalized to the maximum value—comparison with ECFPr. It is a figure which shows the pH dependence of the fluorescence characteristic of purified ECFPr (148R). Graph A: Absorption spectrum normalized to the same region. Graph B: Fluorescence emission spectrum normalized to the same region (λex = 420 nm). Graph C: Fluorescence intensity emitted at 474 nm (Δλ = 6 nm) normalized to the maximum value—comparison with ECFPr. It is a figure which shows the pH dependence of the fluorescence characteristic of purified ECFPr (65S, 148D). Graph A: Absorption spectrum normalized to the same region. Graph B: Fluorescence emission spectrum normalized to the same region (λex = 420 nm). Graph C: Fluorescence intensity emitted at 474 nm (Δλ = 6 nm) normalized to the maximum value—comparison with ECFPr. Graph D: Fluorescence lifetime vs. ECFPr. It is a figure which shows the pH dependence of the fluorescence characteristic of purified ECFPr (65S, 148S). Graph A: Absorption spectrum normalized to the same region. Graph B: Fluorescence emission spectrum normalized to the same region (λex = 420 nm). Graph C: Fluorescence intensity emitted at 474 nm (Δλ = 6 nm) normalized to the maximum value—comparison with ECFPr. Graph D: Fluorescence lifetime vs. ECFPr. It is a figure which shows the pH dependence of the fluorescence characteristic of purified ECFPr (65S, 148E). Graph A: Absorption spectrum normalized to the same region. Graph B: Fluorescence emission spectrum normalized to the same region (λex = 420 nm). Graph C: Fluorescence intensity emitted at 474 nm (Δλ = 6 nm) normalized to the maximum value—comparison with ECFPr. Graph D: Fluorescence lifetime vs. ECFPr. FIG. 5 shows the life span of the fluorescent protein ECFPr, which at least contains the 65S and 148G mutations expressed alone or incorporated into a biosensor—as a function of intracellular pH. BHK (Baby Hamster Kidney) cells were transfected with either a plasmid encoding only ECFPr (65S, 148G) or a pHAKAR biosensor. It is a figure which shows the spectral characteristics of cyan fluorescent protein ECFPr, ECFPr (65S), ECFPr (72A, 145A, 148D) and ECFPr (65S, 72A, 145A, 148D). Absorption spectrum (dotted line) and emission spectrum (solid line) were normalized to the same region (maximum value of chromophore band). The emission spectrum was recorded at an excitation wavelength λex of 420 nm. FIG. 6 shows the photophysical properties of cyan fluorescent protein with or without a 65S mutation. ε: molar extinction coefficient; Q. yield: quantum yield of fluorescence emission; τ _L : longest lifetime value in the fluorescence lifetime distribution; c _L : relative amplitude of the time component of the longest fluorescence lifetime in the fluorescence lifetime distribution; % Rev: Percentage of initial fluorescence intensity loss after abrupt irradiation with a density of 0.2 W / cm ² ; τ _Rev (s): Time constant of initial decay of fluorescence intensity; τ _Irrev (s): 0.2 W / cm ² Exponential time constant of the irreversible loss of fluorescence under long-term irradiation at a density of. It is a figure which shows the fluorescence lifetime distribution of the cyan fluorescent protein which does not contain the 65S mutation. Distributions were obtained through analysis of fluorescence decay of proteins ECFPr, ECFPr (65S), ECFPr (72A, 145A, 148D) and ECFPr (65S, 72A, 145A, 148D) by the maximum entropy method. For each protein, a distribution was established from 6 independent experiments performed at pH 7.4 and 20 ° C. It is a figure which shows the pH dependence of ECFPr spectrum characteristics. (A) Absorption spectrum normalized to unit maximum absorbance, and (B) Emission spectrum normalized to the same region. FIG. 6 shows the emission spectra of cyan fluorescent protein with and without 65S mutation at acidic, neutral and basic pH. (A): ECFPr; (B): ECFPr (72A, 145A, 148D); (C): ECFPr (65S); (D) ECFPr (65S, 72A, 145A, 148D). FIG. 5 shows absorption spectra at acidic, neutral and basic pH of cyan fluorescent protein with or without 65S mutation. (A): ECFPr; (B): ECFPr (72A, 145A, 148D); (C): ECFPr (65S); (D) ECFPr (65S, 72A, 145A, 148D). FIG. 5 shows reversible photobleaching of cyan fluorescent protein with or without a 65S mutation. (A) Reversible fading kinetics performed on agarose beads labeled with purified fluorescent protein. After pre-equilibration in the dark, a rapid and constant illumination with a power density of 0.2 W / cm ² was applied for less than 15 seconds, during which time images were taken every 200 ms. Irradiation was then stopped, and after a minimum of 3 minutes in the dark, a series of fluorescent images were collected to examine reversibility. The continuous line corresponds to the best adjustment based on the model F ^norm = y ₀ + y ₁ t + y ₂ exp (−t / τRev). (B) Reversible photobleaching amplitude of cyan fluorescent protein. FIG. 4 shows photoactivated fluorescence reversion of cyan fluorescent protein ECFPr after transient photobleaching. ECFPr protein was first photobleached using maximum lamp power for less than 1 minute. Next, under different lighting schemes, the return of fluorescence intensity after cessation of irradiation was evaluated: the experimental data was normalized (marker) and the model F ^norm = y ₀ + y ₁ t + y ₂ exp (-t / τ _The best adjustment (continuous line) was evaluated based on _Back ). FIG. 5 shows reversible photobleaching of cyan fluorescent protein with or without 65S mutation in MDCK live cells. The experimental conditions are the same as those used for the experiments with agarose beads. Each curve represents the average of 4 to 6 decays collected from different individual cells. The continuous line is the best adjustment based on the model F ^norm = y ₀ + y ₁ t + y ₂ exp (−t / τ _Rev ). FIG. 5 shows irreversible photobleaching of cyan fluorescent protein with or without 65S mutation in live MDCK cells. (A) A constant irradiation with a power density of 0.2 W / cm ² was applied, and images were taken every 20 seconds during this irradiation. Each curve is the average of 4 to 6 decays collected from individual cells. The continuous line is the best adjustment of the attenuation from the exponential model with the time constant τ _Irrev . (B) Dependence of irreversible fading rate of ECFPr as a function of power density.

In fact, the inventors have discovered that the pH sensitivity of the cyan fluorescent protein ECFP is strongly governed by the nature of specific amino acids, more specifically the amino acids 65 and 148 of the ECFP protein sequence. Thus, the method of the invention comprises the step of introducing a mutation into the cyan fluorescent protein ECFP of the sequence SEQ ID NO: 2, preferably at positions 65 and / or 148 of this sequence. In addition, the introduction of a single mutation can dramatically affect the physicochemical properties, especially the spectral properties, of fluorescent proteins (Espagne et al., 2011; Sawano et al., 2000). The introduction of some mutations at positions 65 and / or 148 of SEQ ID NO: 2 identified by have no negative effect on said properties, and the average fluorescence lifetime of these proteins at neutral pH (tau or τ) can be further increased and pH _1/2 can be decreased.

  “Cyan fluorescent protein” according to the present invention refers to all mutant fluorescent proteins originating from the Aequorea victoria (ECFP) protein sequence, SEQ ID NO: 2, whose absorption spectrum is comprised between 415 and 450 nm. The emission spectrum has a fluorescence maximum value contained in 470 to 490 nm. Preferably, the absorption spectrum of the protein exhibits an absorbance maximum at approximately 435 nm and their emission spectrum exhibits a fluorescence maximum at approximately 476 nm. The absorption spectrum corresponds to the wavelength at which the fluorescent protein is excited, and the emission spectrum corresponds to the wavelength at which the protein emits fluorescence.

Thus, the mutated cyan fluorescent protein according to the present invention has the advantage of exhibiting an absorption and emission spectrum similar to that of the ECFP of SEQ ID NO: 2. Furthermore, the fluorescence intensity (I _f ) of these proteins and their average fluorescence lifetime (tau or τ) are stable and remain higher over a wider pH range.

Thus, the method of the present invention has a fluorescence intensity at acidic pH (I _f ) and its loss of mean fluorescence lifetime (τ) is lower than that of ECFP, in other words less than 50% and 33%, preferably 30% respectively. It makes it possible to obtain lower, even more preferably less than 20% cyan fluorescent protein. Even more preferably, these impairments are zero.

  The life expectancy at neutral pH of these muteins is further enhanced compared to ECFP. Thus, this lifetime is better than 2.5 nanoseconds (ns) and can reach 4.12 ns.

The pH _1/2 of the protein of the present invention is also reduced to a pH _1/2 value lower than that of ECFP, since it is less than 5.6 and can reach pH _1/2 values of 3.4 and even 3.1. Is done.

  Another particular advantage of the present invention lies in the fact that these single mutations can be introduced directly into existing biosensors that are more particularly used in FRET applications. The cyan fluorescent protein according to the present invention can be specifically coupled in a biosensor to an orange fluorescent protein (TagRFP) or a yellow fluorescent protein (YFP type) with reduced pH sensitivity. Therefore, the development of such biosensors has made it possible to study various biological processes under any pH conditions, especially at acidic pH, which has never been possible due to the pH sensitivity of ECFP proteins . In this regard, the present inventors have developed a biosensor with reduced pH sensitivity in which the cyan fluorescent protein of the present invention is coupled to the orange fluorescent protein TagRFP.

  Thus, the present invention meets the requirements of current real-time quantitative imaging methods and allows the use of cyan fluorescent protein under acidic pH conditions.

A first aspect of the invention produces a cyan fluorescent protein exhibiting reduced pH sensitivity, comprising a step in which a single mutation is introduced into a protein sequence comprising the sequence of SEQ ID NO: 2 below. The way is:
VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKQDFKEDGNILGHKLEYNYISHNVYITADKQKGS

  As used herein, “include” or “contain” means that the described element is necessary or essential, but other optional elements may or may not be present. To do.

  Thus, as used herein, a “protein sequence comprising the sequence of SEQ ID NO: 2” refers to any other amino acid such as an N-terminal methionine, signal peptide sequence, or the sequence of SEQ ID NO: 2 at its N-terminal or C-terminal position, or It means that even amino acid sequences that allow protein purification can be included. A person skilled in the art can select an amino acid sequence that enables protein purification.

  Peptide sequences that do not affect the functional properties of the protein, such as those contained in a polyhistidine sequence and / or a protease cleavage site sequence (eg, the sequence is MSYYHHHHHDYDIPTTENLYFQGA SEQ ID NO: 70). Thus, the protein sequence to be mutated can comprise or consist of the sequence of SEQ ID NO: 4.

  According to another preferred embodiment, the sequence of SEQ ID NO: 2 does not contain other amino acids, so that the protein sequence consists only of the sequence of SEQ ID NO: 2.

  Preferably, said mutation is introduced at position 65 or 148 of the sequence of SEQ ID NO: 2.

  According to a particular embodiment of the invention, the method comprises that if the mutation of step a) is introduced at position 148, an additional mutation is introduced at position 65 of the sequence of SEQ ID NO: 2. )including. Conversely, if the mutation of step a) is introduced at position 65, step b) of the method consists in introducing an additional mutation at position 148 of the sequence of SEQ ID NO: 2.

  According to another particular embodiment of the invention, the method consists of steps a) and b) as defined above.

  According to an advantageous embodiment, the method consists only of step a) as defined above.

  Preferably, according to different embodiments of the above method, the amino acid at position 65 is replaced by serine and / or the amino acid at position 148 is replaced by glycine, alanine, serine or glutamic acid. Even more preferably, said amino acid at position 148 is substituted by glycine, alanine or serine. According to another preferred embodiment, the amino acid at position 65 is replaced by serine and the amino acid at position 148 is replaced by glycine, aspartic acid, glutamic acid or serine, preferably glycine, aspartic acid or serine. Even more preferably, the amino acid at position 65 is substituted with serine and the amino acid at position 148 is replaced with glycine.

  Regardless of whether it comprises or consists of step a) or steps a) and b), the method of the present invention produces a cyan fluorescent protein as defined above that exhibits reduced pH sensitivity. It is understood to be possible.

  When the amino acid at position 65 is replaced by serine, the cyan fluorescent protein obtained according to the method of the present invention has an increased quantum yield, simplified fluorescence kinetics, reduced reversible photobleaching, and slowed down. It also shows irreversible photobleaching. As a result of their advantageous photophysical properties, the cyan fluorescent proteins of the present invention containing at least the 65S mutation are particularly useful in live cell imaging applications such as FRET or FLIM type applications.

  According to the method of the present invention, “mutation” means a change in the amino acid sequence of SEQ ID NO: 2 of the protein following modification of the nucleotide sequence of SEQ ID NO: 1 encoding ECFP protein. A mutation according to the invention may be an amino acid addition, deletion or substitution with another amino acid compared to the original protein sequence. Preferably, the mutation is a substitution.

  Methods that allow the introduction of mutations into nucleotide sequences are known to those skilled in the art. Mutations can be introduced, for example, by random or directed mutagenesis, by PCR using degenerate primers, by radiation, or by using mutagens. The technique is described in Sambrook et al., “Molecular Cloning: A laboratory Manual”, 3rd edition, Cold Spring Harbor Laboratory Press, (2001), and Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley & Sons (2011). ). Preferably, the mutation according to the invention is introduced by directed mutagenesis. It is understood that one skilled in the art can use functionally equivalent codons (or nucleotide triplets), ie, codons that encode the same amino acid or a biologically equivalent amino acid, to introduce the mutation. In addition, if one skilled in the art wishes to optimize the expression of the mutant cyan fluorescent protein of the present invention, a website listing the optimal use of codons in various organisms and organelles http: //www.kazusa You can refer to the database on .or.jp / codon /.

  In the context of the present invention, an `` amino acid '' means a natural α-amino acid (e.g. alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ( Cys), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K) ), Methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) And D or L form of valine (Val, V)), as well as all residues of unnatural amino acids.

  Another aspect of the invention relates to a cyan fluorescent protein with reduced pH sensitivity obtained by the above method comprising or consisting of step a) or steps a) and b).

As used herein, “pH sensitivity” or pH dependence according to the present invention refers to the loss of fluorescence intensity (I _f ) of cyan fluorescent protein when the pH of the medium in which the protein is present goes from basic pH to acidic pH and Means a decrease in fluorescence lifetime (tau or τ). The pH sensitivity according to the present invention can be defined by at least one of the two above criteria.

Preferably, the term “pH-sensitive” refers to the loss of fluorescence intensity (I _f ) and fluorescence lifetime (tau or τ) of cyan fluorescent protein when the pH of the medium in which the protein is present goes from basic pH to acidic pH. Refers to a decrease. According to the present invention, the terms impairment, reduction, reduction, attenuation or reduction are synonymous and can be used interchangeably.

The terms fluorescence intensity (I _f ), quantum yield or brightness are interdependent. In the context of the present invention, the term “fluorescence intensity” means the number of photons emitted by a fluorescent protein per unit of volume at a given wavelength and per unit of time. The term “quantum yield” refers to the ratio of the intensity of emitted fluorescence across the emission spectrum and the ratio of the absorption intensity of said protein. With respect to the term “brightness” this refers to the product of the quantum yield and molar absorption coefficient of the protein.

  The “fluorescence intensity” of the fluorescent protein is determined using a spectrofluorometer, such as Fluorolog® 3 (HORIBA Jobin Yvon), and at low concentration conditions, such as

It can be obtained under

And the above formula,
-ε (λ _ex ) and I ₀ (λ _ex ) refer to the molar absorption coefficient of the protein and the intensity of incident light at the excitation wavelength λ _ex , respectively;
-f (λ _em ) represents the fluorescence intensity at the emission wavelength λ _em .

When incorporated into the emission spectrum, this parameter is equal to the quantum yield;
-C is the concentration of the protein;
-l is the length of the optical path in the sample.

  A method that allows the measurement of these parameters is B. Valeur, "Molecular Fluorescence: Principles and Applications", 3rd edition. (2006), described in Wiley-VCH.

In addition, the measurement of fluorescence intensity makes it possible to take into account the following fluctuations:
-Molar absorption coefficient, hence absorption spectrum;
-The possibility of luminescence at the emission wavelength and hence by extension of the emission spectrum when studying proteins under different pH conditions.

  The term “fluorescence lifetime” (Tau, <τ>) means the average time that a fluorescent protein remains in an excited state until it returns to its ground state. This duration is preferably measured in nanoseconds. Here, the fluorescence lifetime is the average lifetime (<τ>), and the fluorescence emission decay I (t) is expressed by the formula:

Obtained by adjusting using the index sum by

It is.

  Here, fluorescence decay is obtained by the unique photon counting method described by O'Connor et al. (1984).

  Among the methods for measuring fluorescence lifetime, we can cite fluorescence lifetime imaging microscopy (FLIM) and time correlated single photon counting (TCSPC).

  The above method is preferably in the temperature range comprised between 0 ° C and 100 ° C, preferably 1 ° C to 90 ° C, 2 ° C to 80 ° C, 3 ° C to 70 ° C, 4 ° C to 60 ° C, 5 ° C to 50 ° C. More preferably 6 ° C to 40 ° C, 7 ° C to 30 ° C, 8 ° C to 25 ° C, 9 ° C to 24 ° C, 10 ° C to 23 ° C, even more preferably 11 ° C to 22 ° C, It can implement in the range contained in 12 to 21 degreeC. Advantageously, the temperature range is 20 ° C. ± 0.1 ° C.

  The basic pH means a pH value included in 7 to 14, and the acidic pH means a pH value included in 0 to 7. Thus, the pH sensitivity according to the present invention is over a pH range of 0 to 14, preferably over a pH range of pH 1 to pH 13, pH 2 to pH 12, more preferably over a range of pH 2.5 to pH 11, pH 3 to 10, pH 4 to pH 9. Even more preferably, it is studied over a range of pH 5 to pH 8 and pH 5.5 to pH 7.5. Advantageously, the pH tested is from pH 5.5 to pH 7.4. The above depletion, reduction or change is always measured according to the invention between the most basic pH and the most acidic pH.

“Reduced pH sensitivity” refers to a decrease in the fluorescence intensity (I _f ) of a protein obtained by the method of the present invention when the pH of the medium in which the protein is present changes from a basic pH to an acidic pH. Means less than%, preferably less than 33%, even more preferably less than 30%, 25%, 20%, 15%, 10%, 5%, advantageously equal to 0%. More specifically, when the pH is from pH 7.4 to pH 5.5, the loss of fluorescence intensity is less than 50%, preferably less than 33%, and even more preferably 30%, 25%, 20%, Less than 15%, 10%, 5%, advantageously equal to 0%.

  The term “reduced pH sensitivity” means that the loss of fluorescence lifetime (tau or τ) of a protein obtained by the method of the present invention is such that when the pH of the medium in which the protein is present goes from basic pH to acidic pH. It can also mean less than 33%, preferably less than 32%, even more preferably less than 30%, 25%, 20%, 15%, 10%, 5%, advantageously equal to 0%. . More specifically, when the pH goes from pH 7.4 to pH 5.5, the loss of fluorescence lifetime is less than 33%, preferably less than 32%, even more preferably 30%, 25%, 20%, Less than 15%, 10%, 5%, advantageously equal to 0%.

  When at least one of the two criteria defined above is met, the present invention reduces the pH sensitivity of the cyan fluorescent protein.

  Preferably, the pH sensitivity of the cyan fluorescent protein is reduced if the two criteria defined above are met.

In addition to these criteria, cyan fluorescent proteins with “reduced pH sensitivity” sometimes show a decrease in their half-transition pH (pH _1/2 ) relative to the ECFP pH _1/2 value. it can. Accordingly, the reduction in pH _1/2 is at least 0.1, 0.2, 0.3, 0.4 pH units, preferably at least 0.5, 1, 1.5, ₂ pH units, and even more preferably at least 2.2 pH units.

As used herein, “pH _1/2 ” according to the present invention means the pH value at which the sum of the fluorescence intensity at 474 nm of the protein at the most basic pH and the most acidic pH as defined above is halved. To do. The fluorescence intensity is measured by the method defined above. Preferably, pH _1/2 is the pH value at which the total fluorescence intensity at 474 nm of the protein at pH 7.4 and pH 2.5 is halved. An equivalent method for measuring the semi-transition pH of a fluorescent protein is the total amount released by the protein by calculating the area under the fluorescence emission spectrum obtained by excitation at 420 nm at each test pH value. The purpose is to measure the intensity of fluorescence. This total intensity is then corrected from the absorbance at 420 nm. The semi-transition period can also be estimated from variations as a function of the pH of the corrected total intensity thus obtained by similar calculation methods (pH values such as the sum of these total intensities are the most basic pH And the most acidic is halved).

  Furthermore, these proteins can also show a positive increase in their fluorescence lifetime (tau or τ) at pH 7.4 compared to the ECFP lifetime. Accordingly, the increase in τ value is at least 0.1 ns, preferably at least 0.5 ns, 1 ns, and even more preferably at least 1.5 ns.

  Nevertheless, the “reduced pH sensitivity” property should not be compared to what is known as thermodynamic or kinetic stability, more commonly “stability”. In fact, the “stability” of a protein is characterized by the retention of the native structure of this protein within a given range of external physicochemical conditions (temperature, pressure, etc.) because it destroys the protein structure This is a complex concept that can be difficult to evaluate because it depends not only on the external conditions used for the protein, but also on the parameters selected to evaluate the denaturation state of this protein.

  The criteria according to the present invention describing cyan fluorescent protein follow the following rule: ECFP (mutant amino acid number-name of the introduced amino acid).

  Amino acid numbering according to the present invention is based on the valine amino acid sequence of ECFP SEQ ID NO: 2 rather than the conventional one based on the N-terminal methionine amino acid. Thus, the sequence of SEQ ID NO: 2 serves as a reference for assigning the number or position of the mutated amino acid, and the name of the introduced amino acid is the same as the sequence of SEQ ID NO: 2, as further described. It can be determined by performing an optimal alignment with that of the cyan fluorescent protein obtained by the method of the invention.

  Examples of cyan fluorescent protein with reduced pH sensitivity according to the present invention are SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 58, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 60, SEQ ID NO: 72, A protein sequence selected from the group consisting of the sequences of SEQ ID NO: 74 and SEQ ID NO: 76 (see Table 1 below) is included or is not limited thereto.

  According to a preferred embodiment of the present invention, the cyan fluorescent protein according to the present invention is not limited to a protein sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 60, SEQ ID NO: 74 and SEQ ID NO: 76. Contain or consist of.

  According to another advantageous embodiment, the cyan fluorescent protein according to the invention comprises or consists of a protein sequence of the sequence SEQ ID NO: 12.

  The invention also relates to a nucleic acid encoding a cyan fluorescent protein as defined above.

  Does the “nucleic acid” or nucleotide sequence comprise non-natural nucleotides that can correspond to both single-stranded or double-stranded DNA selected from cDNA, genomic DNA and plasmid DNA and the transcription products of said DNA? It means the exact strand of modified or unmodified nucleotides that makes it possible to define a nucleic acid fragment or region that does not contain.

  Examples of nucleic acids according to the present invention are SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 57, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 59, SEQ ID NO: 71, SEQ ID NO: 73 and SEQ ID NO: 75 (see below It includes, or is not limited to, a nucleotide sequence selected from the group consisting of the sequences in Table 1 (see Table 1).

  According to a preferred embodiment of the invention, the nucleic acid comprises or consists of a nucleotide sequence selected from the group consisting of the sequence of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 59, SEQ ID NO: 73 and SEQ ID NO: 75. .

  According to another advantageous embodiment, the nucleic acid according to the invention comprises or consists of the nucleotide sequence of the sequence SEQ ID NO: 11.

  The nucleic acids defined above may or may not contain a start codon at the 5 ′ end and / or a stop codon at the 3 ′ end of their sequences. Initiation codons include, but are not limited to, codons (or trinucleotides or nucleotide triplets) ATG, AUG, TTG, UUG, GTG, GUG, CTG and CUG, based on the host cell in which the nucleic acid to be translated is present. Can be selected by those skilled in the art. Stop codons specifically include the codons TAG, UAG, TAA, UAA, TGA and UGA, which can be selected by one skilled in the art based on the host cell in which the nucleic acid to be translated is present.

  Advantageously, these nucleic acids do not include a start codon and / or a stop codon when the cyan fluorescent protein they encode is fused directly or indirectly to another protein.

  These nucleic acids may also contain, at their 5 ′ and / or 3 ′ ends, nucleotide sequences that encode signal peptides and / or amino acid sequences that allow purification of the proteins they encode. it can. The latter can be selected by those skilled in the art from nucleotides that do not affect the functional properties of the protein, e.g. polyhistidine sequences and / or protease cleavage site sequences (e.g. the sequence is ATGTCGTACT ACCATCACCA TCACCATCAC GATTACGATA TCCCAACGAC CGAAAACCTG TATTTTCAGG GCGCC SEQ ID NO: 69).

  According to another embodiment of the present invention, the method comprises 9G, 11I, 19E, 26R, 68L, 72A, 87V, 145A, 164H, 167A, 172T in addition to step a), or step a) and step b). , 175G, 194I and 206K, wherein at least one other mutation is introduced into the sequence of SEQ ID NO: 2 and is performed either before or after step a) and / or step b) including c).

  Thus, a “cyan fluorescent protein” according to the present invention can have one or several auxiliary (or additional) mutations at positions other than positions 65 and 148, provided that these mutations But only if it allows preservation of the absorption and emission spectra of the protein of the invention described above. Such conservative mutations are known to those skilled in the art and are among the mutations 9G, 11I, 19E, 26R, 68L, 72A, 87V, 145A, 164H, 167A, 172T, 175G, 194I and 206K, Preferably, the mutations 26R, 72A, 145A, 164H and 206K can be selected without limitation. These auxiliary mutations can be further introduced in the same way either before or after the 65 and / or 148 mutations characteristic of the present invention. Thus, the method of the invention comprising or consisting of steps a), b) and c) enables the production of a cyan fluorescent protein as defined above that exhibits reduced pH sensitivity.

  Thus, the amino acid sequence of the mutant cyan fluorescent protein is thus at least about 60%, 65%, 70%, 75%, 80% with the sequence of ECFP SEQ ID NO: 2, either adjacent or not adjacent. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity. Preferably, the amino acid sequence according to the present invention exhibits at least about 85%, advantageously at least about 90% identity with the sequence of SEQ ID NO: 2, adjacent or not. Even more preferably, the amino acid sequence according to the present invention has at least about 95% identity, advantageously at least about 97%, even more preferably at least about Shows 99% identity.

  The above percentage identity can be determined by performing an optimal alignment of the amino acid sequences to be compared (here with the sequence of SEQ ID NO: 2), this percentage is purely statistical and between the two sequences Differences are distributed over their entire length. This alignment is performed using algorithms known to those skilled in the art, such as the global alignment and computerized application of Needleman and Wunsch (J. Mol. Biol., 1970, 48: 443) or simply by inspection. be able to. The best alignment (ie the one that produces the highest percentage identity) is then selected. The percent identity between two amino acid sequences is determined by the addition or deletion of the compared amino acid sequence compared to a reference sequence (here, the sequence of SEQ ID NO: 2) for optimal alignment of these two sequences. It is determined by comparing these two sequences aligned in an optimal manner that can be included. The percent identity is determined by determining the number of identical positions where amino acid residues are identical between the two sequences, dividing the number of identical positions by the total number of positions and multiplying the result obtained by 100. Calculated by obtaining the percent identity between two sequences.

  Preferably 1 to 20 auxiliary mutations, preferably 1 to 10 mutations, even more preferably 1 to 5 mutations, advantageously 1 to 2 mutations of SEQ ID NO: 2. Can be introduced into the sequence.

  According to a preferred embodiment of the present invention, the additional mutations introduced into the sequence of SEQ ID NO: 2 are the mutations 9G, 11I, 19E, 26R, 68L, 72A, 87V, 145A, 164H, 167A, 172T, 175G, 194I And 206K alone. Even more preferably, these additional mutations are selected alone from among mutations 26R, 72A, 145A, 164H and 206K.

  Examples of proteins that can be generated in this way are SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 62, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 64, SEQ ID NO: 28, SEQ ID NO: A protein sequence selected from the group consisting of the sequence of SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 66, SEQ ID NO: 34, SEQ ID NO: 80 and SEQ ID NO: 82 (see Table 1 below) is not limited. Contains or consists of.

  According to a preferred embodiment, the cyan fluorescent protein of the present invention comprises SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 62, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 64, SEQ ID NO: 28, SEQ ID NO: 30, A protein sequence selected from the group consisting of the sequence of SEQ ID NO: 32, SEQ ID NO: 66, SEQ ID NO: 34, SEQ ID NO: 80 and SEQ ID NO: 82 is included or consists in a non-limiting manner.

  According to an even more preferred embodiment, the cyan fluorescent protein according to the invention comprises SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 62, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 64, SEQ ID NO: 28, SEQ ID NO: 30, comprises or consists of a protein sequence selected from the group consisting of the sequences of SEQ ID NO: 32, SEQ ID NO: 66, SEQ ID NO: 80, and SEQ ID NO: 82.

  Even more preferably, the cyan fluorescent protein of the invention comprises SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 64, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 66, SEQ ID NO: 80 and A protein sequence selected from the group consisting of the sequence of SEQ ID NO: 82 is included or consists in a non-limiting manner.

  According to an even more advantageous embodiment, the cyan fluorescent protein of the invention comprises, but is not limited to, a protein sequence selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 64 It consists of it.

  According to another advantageous embodiment of the invention, the cyan fluorescent protein of the invention has a non-limiting protein sequence selected from the group consisting of the sequences SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 and SEQ ID NO: 66. Contain or consist of.

  According to another advantageous embodiment, the cyan fluorescent protein of the invention comprises or consists of the protein sequence of SEQ ID NO: 80 or SEQ ID NO: 82.

  The present invention also relates to a nucleic acid encoding the above-described cyan fluorescent protein.

  Thus, the nucleic acid encoding the protein or nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85% with the sequence of SEQ ID NO: 1 of ECFP, either contiguously or not. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity. Preferably, the nucleotide sequence according to the invention exhibits at least about 85%, advantageously at least about 90% identity with the sequence of SEQ ID NO: 1, either contiguously or not. Even more preferably, the nucleotide sequence according to the present invention is at least about 95% identity, advantageously at least about 97%, even more preferably at least about Shows 99% identity.

  The above percent identity can be determined by performing an optimal alignment of the nucleotide sequences to be compared (here with the sequence of SEQ ID NO: 1), this percentage is purely statistical and between the two sequences Differences are distributed over their entire length. This alignment is performed using algorithms known to those skilled in the art, such as the global alignment and computerized application of Needleman and Wunsch (J. Mol. Biol., 1970, 48: 443) or simply by inspection. be able to. The best alignment (ie the one that produces the highest percentage identity) is then selected. The percent identity between two nucleotide sequences is the addition or deletion of the compared nucleotide sequence compared to a reference sequence (in this case the sequence of SEQ ID NO: 1) for optimal alignment of these two sequences. By comparing these two sequences aligned in an optimal manner. The percent identity determines the number of identical positions where the nucleic acid is identical between two sequences, divides the number of identical positions by the total number of positions, and multiplies the result obtained by 100 to obtain these two sequences. Calculated by obtaining the percent identity between.

  Examples of nucleic acids encoding proteins of the invention that exhibit additional mutations are SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 61, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 63, A nucleotide sequence selected from the group consisting of the sequences of SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 65, SEQ ID NO: 33, SEQ ID NO: 79 and SEQ ID NO: 81 (see Table 1 below) Including, but not limited to.

  According to a preferred embodiment, the nucleic acid encoding the cyan fluorescent protein according to the invention is SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 61, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 63, SEQ ID NO: 27, The nucleotide sequence selected from the group consisting of the sequence of SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 65, SEQ ID NO: 33, SEQ ID NO: 79 and SEQ ID NO: 81 is included or consists in a non-limiting manner.

  According to a more preferred embodiment, the nucleic acid according to the invention comprises SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 61, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 63, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NO: 65, SEQ ID NO: 79 and SEQ ID NO: 81.

  Even more preferably, the nucleic acid according to the present invention comprises SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 63, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 65, SEQ ID NO: 79 and SEQ ID NO: 81. A nucleotide sequence selected from the group consisting of:

  According to an even more advantageous embodiment, the nucleic acid of the invention comprises or consists in a non-limiting manner a nucleotide sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 and SEQ ID NO: 63.

  According to another advantageous embodiment of the invention, the nucleic acid of the invention comprises, but is not limited to, a nucleotide sequence selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 and SEQ ID NO: 65. It consists of it.

  According to another advantageous embodiment, the nucleic acid according to the invention comprises or consists of the nucleotide sequence of SEQ ID NO: 79 or SEQ ID NO: 81.

  Another aspect of the present invention relates to a recombinant vector comprising a nucleic acid encoding a mutant cyan fluorescent protein according to the present invention as defined above. Thus, the nucleic acid can be inserted into a replicable vector for the purpose of amplifying the nucleic acid or expressing the cyan fluorescent protein according to the invention. Vectors according to the present invention include cloning vectors that allow nucleic acid amplification and expression vectors that allow protein expression, and these vectors are well known to those of skill in the art. Such vectors include, but are not limited to, plasmids, cosmids, YACS, viruses (adenoviruses, retroviruses, episomal EBV) and phage vectors. One skilled in the art can further use any other suitable vector that allows expression of the cyan fluorescent protein of the present invention.

  Methods for inserting nucleic acids into such vectors are known to those skilled in the art. In general, nucleic acids are inserted into at least one suitable endonuclease restriction site using techniques known in the art (e.g., the techniques described in Sambrook et al. (2001) and Ausubel et al. (2011)). reference).

  Further included in the recombinant vector of the invention is a nucleotide sequence that allows transcription of the nucleic acid of the invention, expression and / or purification of the cyan fluorescent protein of the invention. These sequences generally and without limitation include one or several peptide signal sequences, origins of replication, one or several selectable gene markers, amplification elements, promoters, transcription termination sequences, and possibly protein Sequences that allow purification are included. Insertion of such sequences into the vector can be performed by standard ligation techniques known to those skilled in the art.

  Depending on the origin of replication chosen, the cloning or expression vector can replicate in one or more host cells. Thus, the origin of replication of plasmid pBR322 is compatible with the majority of gram-negative bacteria, that of plasmid 2μ is specific for yeast, and various viral origins of replication (SV40, polyome, adenovirus, VSV or BPV) are suckling. Useful for cloning vectors in animal cells.

  Depending on the promoter chosen, the nucleic acid can be transcribed and the corresponding protein expressed in one or more host cells. Thus, promoters T7, Lac, trp, tac, λPL are specific for bacteria of the genus E. coli; promoters PHO5, GAP, TPI1, ADH are compatible with yeast; promoters polyhedrin and P10 and Their equivalents are used in insect cells; finally, the promoter CMV, MT1, derived SV40, SRα, retrovirus, and heat shock protein gene promoters are compatible with mammalian cells.

  Among the selectable marker genes typically included in cloning or expression vectors, the following can be cited without limitation: (a) antibiotics or toxins (e.g., neomycin, zeocin, hygromycin, ampicillin, Encodes a gene that confers resistance to kanamycin, tetracycline, chloramphenicol), and (b) a gene that enables compensation for auxotrophic defects (e.g., the dihydrofolate reductase DHFR that enables resistance to methotrexate) Gene, or S. pombe TPI gene).

  Nucleotide sequences that allow protein purification include, but are not limited to, histidine sequences (histidine tag or Hisx6), FLAG sequences, and GST sequences. A protease cleavage sequence such as TEV may then be present to subsequently suppress the purified sequence.

  According to a preferred embodiment of the present invention, a nucleotide sequence encoding the nucleotide sequence of six histidines and the protease cleavage sequence TEV, more particularly the amino acid sequence MSYYHHHHHHDYDIPTTENLYFQGA (SEQ ID NO: 70) is present in the recombinant vector of the present invention.

  According to a particularly preferred embodiment of the invention, the expression vector of the invention is the vector pPROEX ™ HTa (GibcoBRL).

  Methods for connecting nucleic acids according to the present invention to the above additional sequences are known to those skilled in the art.

  The invention also relates to a host cell transformed with a nucleic acid encoding said protein or with a recombinant vector comprising said nucleic acid.

  As used herein, the terms “host cell”, “cell” and “cell line” can be used interchangeably. All these terms include their progeny, which includes all subsequent generations. In view of intentional or accidental mutations, it is understood that not all progenies are necessarily the same.

  As used herein, a “host cell” is capable of replicating a nucleic acid encoding a mutant fluorescent protein according to the present invention or the aforementioned recombinant vector, and thus expressing the mutant fluorescent protein of the present invention. Refers to prokaryotic or eukaryotic cells capable of A host cell can be “transfected” or “transformed” by methods known to those of skill in the art in which the nucleic acid or the vector is transferred or introduced into the host cell. Examples of such methods include, but are not limited to, electroporation, lipofection, calcium phosphate transfection, DEAE-dextran transfection, microinjection and particle bombardment.

  Host cells include, but are not limited to, bacteria, yeast, fungi or any other higher eukaryotic cell. Thus, particularly through the American Type Culture Collection (ATCC) (www.ATCC.org), one skilled in the art can select an appropriate host cell from among many available cell lines. Examples of host cells include, but are not limited to, microorganisms such as Escherichia (e.g., Escherichia coli RR1, LE392, B, X1776, W3110, DH5 alpha, JM109, KC8), Serratia, Pseudomonas. (Pseudomonas), Erwinia, Methylobacterium, Rhodobacter, Salmonella or Zymomonas Gram-negative bacteria, Corynebacterium, Brevi Gram-positive bacteria of the genus Brevibacterium, Bacillus, Arthrobacter or Streptomyces, Saccharomyces yeast, Aspergillus, Neurospora ( Neurospora), cells from Fusarium and Trichoderma fungi, HEK293, NIH3T3, Jurkat, MEF, Vero, HeLa, CHO W138, BHK, include COS-7, MDCK, C127, Saos, PC12, Sf9, animal cells, and insect cells including HKG, Sf21, Hi Five (TM) or silkworm (Bombyx mori). The use of insect cells is described in particular in the manual “Baculovirus Expression vectors, A Laboratory Manual”, David R. O'Reilly et al., Oxford University Press, USA, (1992).

  When the host cell is transformed with the above-described recombinant vector of the present invention, the selection of the host cell depends on the selection of the vector and the selected use, ie cloning of the nucleic acid or mutant cyanide of the present invention. It can be determined according to the expression of the fluorescent protein.

  Another aspect of the invention relates to a method for expressing and purifying the mutant cyan fluorescent protein of the invention. By this method, the above host cells are cultured in a suitable culture medium under conditions that allow expression of the protein of the invention. One skilled in the art can use any conventional method that allows for isolation and purification of the protein. For example, if the protein is expressed in a soluble form in the host cell, the latter is recovered by centrifugation and suspended in a buffer, and then cell extraction by disrupting the cell, for example by an ultrasonic homogenizer. A thing is obtained. From the supernatant obtained by centrifugation of this extract, a purified sample is obtained using conventional methods or combinations of conventional methods for isolating and purifying the proteins of the invention. These methods include solvent extraction, ammonium sulfate release, desalting, organic solvent precipitation, gel filtration, preparative electrophoresis, isoelectric focusing, ultrafiltration, a number of chromatographic methods such as ion exchange chromatography ( For example, anionic using a resin such as diethylaminoethyl (DEAE) Sepharose or cationic using a resin such as S-Sepharose (Pharmacia), hydrophobic chromatography (for example, using a resin such as butyl Sepharose or phenyl Sepharose), Examples include, but are not limited to, affinity chromatography with antibodies, adsorption chromatography, chromatographic localization, high performance liquid chromatography (HPLC) and reverse phase HPLC.

  In addition, if the recombinant vector contains a nucleotide sequence that is fused to a cleavage sequence by a protease, allowing purification of the protein, the protein produced is a protease specific for the cleavage sequence (thrombin, trypsin, protease It can be recovered through processing by TEV etc.).

  Another aspect of the present invention relates to a biosensor comprising a cyan fluorescent protein according to the present invention.

  As used herein, a “biosensor” includes a biosensitive sensor that is covalently or non-covalently linked to another molecule, and that signals a biological response to an electrical, chemical, physical, photophysical or photochemical signal. It should be understood as a molecule to be converted to. A “biosensitive sensor” according to the present invention is a natural or synthetic molecule that, when expressed in a host cell as defined above, is an ion, sugar, enzyme, nucleic acid, antibody, cofactor or naturally occurring. Allows detection of the presence of an analyte such as a protein that is or has been modified (eg by glycosylation or phosphorylation) and / or determination of its concentration or activity. Thus, the interaction between the analyte present in the cell and the sensor results in a structural rearrangement of the biosensor, which is reflected by the signal described above. The sensor may be a sugar, lipid, protein or nucleic acid, for example. Preferably, the sensor is a peptide molecule.

In accordance with a preferred embodiment of the present invention, biosensitive sensors also include, but are not limited to, peptides such as melittin, hybrid polypeptides (ie, polypeptides resulting from the fusion of at least two proteins), antibodies, enzymes or enzyme substrates. Not. Among the hybrid polypeptides, enzymes fused to the binding site (for example, calmodulin fused to peptide M13, or GTPase fused to a domain capable of recognizing its active conformation). Can be quoted. Examples of sensitive biosensors include peptide sequences that are protein kinase substrates, peptide sequences that are protease substrates, cAMP binding domains, phosphorylated amino acid binding domains (FHA), glutamate binding domains (YbeJ), sucrose binding domains (e.g., maltose MBPs that bind to), as well as Ca ²⁺ binding domains such as troponin C and fragments thereof.

  Thus, the biosensor according to the present invention can enable the study of cell metabolism or cell signaling events.

  In the context of the present invention, a biosensor includes a cyan fluorescent protein that exhibits reduced pH sensitivity, linked to the sensitive biosensor by covalent or non-covalent bonds. Thus, when this biosensor is expressed in a host cell, the interaction between the analyte present in the cell and the sensor results in structural rearrangement of the biosensor, which is the fluorescence to which it is fused Reflected by modification of protein fluorescence. As used herein, “modification of fluorescence emission” is a variation in fluorescence intensity emitted by the protein, wherein the variation is proportional to the concentration or activity of the analyte or reflects the presence of the analyte. It means a variation characterized by The fluorescence intensity can be measured as described previously.

  According to a preferred embodiment, the biosensor of the invention further comprises a fluorescent protein whose absorption spectrum partially overlaps the emission spectrum of the cyan protein of the biosensor.

  According to a particular embodiment, the fluorescent protein whose absorption spectrum partially overlaps with the emission spectrum of cyan protein is directly linked to the biosensor. As used herein, “directly linked” means that the protein is linked to a biosensitive sensor of the biosensor by covalent or non-covalent bonds.

  According to another particular embodiment, a fluorescent protein whose absorption spectrum partially overlaps with the emission spectrum of cyan protein is indirectly linked to the biosensor. As used herein, “indirectly linked” means that the protein is linked to the biosensitive sensor through a molecule, eg, through a non-covalent bond between the molecule and the biosensor biosensitive sensor. means.

  Preferably, the fluorescent protein whose absorption spectrum partially overlaps with the emission spectrum of the cyan protein of the invention is 495-600 nm, preferably 500-590 nm, even more preferably 510-580 nm, even more advantageously Indicates the maximum absorbance contained in 520 to 570 nm and 550 to 570 nm. Examples of fluorescent proteins that can be fused to the proteins of the invention in this way through a biosensitive sensor include, but are not limited to, yellow fluorescent proteins such as YFP, Topaz, EYFP, YPET, SYFP2, Citrine, Venus , Cp-Venus, fluorescent orange proteins such as Kusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato, dTomato-Tandem, DsRed and its variants (DsRed2, DsRed-Express (T1), DsRed-Express2, DsRed-Max , DsRed-Monomer), TagRFP and TagRFP-T, red fluorescent proteins such as mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry, eqFP611, tdRFP611, HcRed1, mRaspberry, and far red emitting fluorescent proteins such as tdRFP639 , MKate, mKate2, Katushka, tdKatushka, HcRed-Tandem, mPlum and AQ143 (Day et al., 2009). Preferably, the fluorescent protein whose absorption spectrum partially overlaps with the emission spectrum of the cyan protein of the present invention is selected from the yellow or orange fluorescent proteins described above.

  In a particularly advantageous manner, a fluorescent protein whose absorption spectrum partially overlaps the emission spectrum of the cyan protein of the invention exhibits itself a reduced pH sensitivity. The protein can then be selected from the proteins Citrine, Venus, cp-Venus, TagRFP and TagRFP-T, preferably TagRFP and TagRFP-T, and even more preferably the protein is TagRFP Yes (Merzlyak et al., 2007).

  According to a particularly advantageous embodiment of the invention, the biosensor of the invention comprises a TagRFP fluorescent protein in addition to the cyan fluorescent protein of the invention whose sequence comprises at least the 65S and 148G mutations. According to a preferred embodiment, said cyan fluorescent protein comprises or consists of a protein sequence selected from the group consisting of the sequence of SEQ ID NO: 12, SEQ ID NO: 80 or SEQ ID NO: 82, even more preferably the protein sequence of SEQ ID NO: 82 .

  The fusion of the biosensitive sensor to the fluorescent protein for the construction of the biosensor of the present invention is performed using genetic engineering, enzymatic or chemical coupling techniques known to those skilled in the art. Such techniques are described in Sambrook et al. (2001) and Ausubel et al. (2011).

  According to a particularly advantageous embodiment of the invention, the biosensor is generated by mutating an existing biosensor by the method of the invention, ie by introducing at least one specific mutation that reduces pH sensitivity. be able to. Other existing biosensors known to those skilled in the art include, but are not limited to, biosensors Epac, Epac2, ECFP-YbeJ-Venus, ECFP-MBP-EYFP, CFP-TnC-Citrine, Aktus, Atomic, Reference may be made to Erkus, Phocus, Picchu, AKAR, AKAR1 (Zhang et al., 2001), AKAR2 (Zhang et al., 2005), AKAR2.2 (Dunn et al., 2006), AKAR3, BKAR, CKAR and DKAR. Preferably, the mutated biosensor is an AKAR type biosensor, and even more preferably the biosensor is AKAR2.2.

  According to an advantageous embodiment, the 65S and 148G mutations are introduced into the biosensor by the method of the invention. Thus, when the 65S and 148G mutations are introduced into the AKAR2.2 biosensor, the latter includes a cyan fluorescent protein comprising the sequence of SEQ ID NO: 82. Even more preferably, the 65S and 148G mutations are introduced into the AKAR2.2 biosensor by the method of the present invention, and the Citrine expressed thereby is classical genetic engineering, enzymatic or chemical coupling techniques known to those skilled in the art. Exchanged for TagRFP protein, whereby the biosensor comprises a cyan fluorescent protein comprising the sequence of SEQ ID NO: 82 and a TagRFP orange fluorescent protein.

  For more detailed information on biosensors, one skilled in the art can refer to publications by M.C. Morris (2010) and Frommer et al. (2009).

  The biosensors of the present invention can be used by the following techniques, more preferably they are FRET and / or FLIM according to the present invention.

  Another aspect of the invention relates to the use of the products of the invention (particularly the mutant fluorescent proteins defined above, nucleic acids encoding these proteins, recombinant vectors, host cells and biosensors).

  These include, but are not limited to, flow cytometry (FACS), conventional imaging methods such as photon or confocal microscopy, real-time quantitative imaging methods such as fluorescence correlation spectroscopy (FCS) and variations thereof (e.g. FCCS), Forster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), fluorescence lifetime imaging microscopy (FLIM), fluorescence redistribution after photobleaching (FRAP), fluorescence loss induced by photobleaching ( FLIP), time correlated single photon counting (TCSPC), anisotropic and fluorescence depolarization, photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), stimulated emission suppression mirror Inspection (STED) is included. This includes high-throughput detection methods such as high content screening, high-throughput microscopy, and conventional or ultra-high-throughput microfluidic methods.

  The most preferred imaging methods used according to the present invention are FRET and FLIM.

  As used herein, the term “Forster Resonance Energy Transfer” (FRET) refers to a non-radiative transition of excitation energy from a photon to a “acceptor” fluorescent molecule that originates from the fluorescent molecule “donor”, and Only possible when the “donor” is close enough to the “acceptor” molecule, ie, at a distance of 10-100 km according to the molecular geometry and observation system (Heyduk (2002), Truong et al. (2001), Issad et al. (2003), Boute et al. (2002)). This method reduces the fluorescence of the photon “donor” (for example by measuring the intensity or lifetime of the fluorescence of the “donor”) or increases the fluorescence of the “acceptor” (for example, the fluorescence intensity of the “acceptor”). Allows quantification of either) Any method derived from FRET also applies to the present invention. In the case of FRET, the mutant cyan fluorescent protein according to the invention is preferably used as a “donor” for photons.

  `` Bioluminescence resonance energy transfer '' or BRET is that the energy of the `` donor '' originates from a bioluminescent molecule that is excited in the presence of an enzyme (e.g. luciferase) to emit a photon, e.g. luciferin (e.g. coelenterazine). And different from FRET. These photons are then transferred to “acceptor” fluorescent molecules of the GFP type that emit fluorescence if the proximity conditions and geometry for energy transfer are met. Thus, the mutant fluorescent protein according to the present invention must be used as an “acceptor” fluorescent molecule in this type of application.

  FLIM (or fluorescence lifetime imaging microscopy) is a technique that allows measurement of the fluorescence decay of a fluorescent molecule and quantification of the fluorescence lifetime of this molecule. This technique can be used alone or in combination with FRET, especially for the localization of protein-protein interactions or for the study of cell signaling. Any method derived from FLIM, such as tomographic FLIM, multiplex FLIM, multiwell plate automated FLIM or confocal endoscopic FLIM is included in the present invention. Where FLIM technology is used in the present invention, preferably the mutant cyan fluorescent protein is a photon donor.

  For any further details regarding the foregoing techniques, one skilled in the art can refer to articles by Day et al. (2009), Trugnan et al. (2004) and Kumar et al. (2011).

  Particular embodiments relate to the use of the products of the invention in screening methods for compounds and / or cells. Preferably, the screening methods are high-throughput detection methods such as high content screening, flow cytometry, high-throughput microscopy, microfluidic methods (conventional or ultra-high throughput) and plate reader assays.

  Another particular embodiment relates to the use of said product in a toxicology, genotoxicity or environmental contamination detection test performed in solution, more particularly in solution from a sample, biological extract, cell, tissue or organism. .

  The invention will be better understood in the light of the following examples.

(Example)
A-Materials and methods
I. Cloning of Cyan Fluorescent Protein An ECFP (pHis-ECFP) expression plasmid was constructed from the pECFPN1 vector (Clontech) as follows: The entire ECFP sequence (SEQ ID NO: 1) of the pECFPN1 vector is the sequence of SEQ ID NO: 35. PCR was carried out using 5′-AAGGCGCCGTGAGCAAGGGCGAGGAGCTG-3 ′ (forward primer) and 5′-TTAAGCTTACTTGTACAGCTCGTCCATGCC-3 ′ (reverse primer) of SEQ ID NO: 36.

  The resulting PCR product was then digested with HindIII and EheI, ligated into the pPROEX-HTa expression vector (GibcoBRL), and then examined by restriction mapping and sequencing. To facilitate purification of the resulting recombinant protein (herein SEQ ID NO: 4), the sequence MSYYHHHHHDYDIPTTENLYFQGA (SEQ ID NO: 70) of the pPROEX-HTa vector contains 6 histidine and protease TEV cleavage sites. Cloning was performed so that it was inserted at the end. The fluorescence properties of ECFP are not affected by this sequence.

II. Methods enabling reduction of pH sensitivity of cyan fluorescent protein
II.1.Directed mutagenesis
A single mutation was introduced into the cyan fluorescent protein by directed mutagenesis according to the “QuikChange mutagenesis” protocol from Stratagene. Specific primers were designed for each mutation (see Table 2).

  Thus, the 148G monomer mutation was introduced using the 148Gf (SEQ ID NO: 37) and 148Gr (SEQ ID NO: 38) primers; the 148S monomer mutation was used with the 148Sf (SEQ ID NO: 39) and 148Sr (SEQ ID NO: 40) primers. The 65S monomer mutation was introduced through the 65Sf (SEQ ID NO: 49) and 65Sr (SEQ ID NO: 50) primers.

  Briefly, the reaction conditions used were as follows: 39 μL of water, 1 μL of plasmid vector (10 ng / μL) expressing the cyan fluorescent protein to be mutated (e.g., pHis-ECFP), suddenly introduced 1.5 μL (100 ng / μL) of each forward and reverse primer specific for the mutation, 1 μL dNTP (10 mM), 5 μL reaction buffer 10 × and 1 μL DNA polymerase PfuUltra HF (2.5 U / μL) were added. Amplification of the mutated nucleotide sequence was then obtained by exposing this mixture to the following conditions of the thermocycler: initial cycle of 2 minutes at 95 ° C, followed by 30 seconds at 95 ° C for each cycle, 58 ° C. 18 amplification cycles of 30 seconds at 72 ° C and 10 minutes at 72 ° C, and finally 10 minutes at 72 ° C Finally, the presence of the mutation in the PCR product was checked by digestion with DpnI enzyme (2.5 U; 1 hour at 37 ° C.) and DNA sequencing.

By the same method of directed mutagenesis, the following additional mutations were introduced before or after the aforementioned mutations:
-Using primers of sequence 72A, SEQ ID NO 51 and SEQ ID 52;
-Using primers of the sequences 145A, SEQ ID NO 53 and SEQ ID NO 54;
-Use primers of 206K, SEQ ID NO: 55 and SEQ ID NO: 56.

II.2. Generation and purification of cyan fluorescent protein mutants Generation and purification of cyan fluorescent protein mutants were performed according to the following protocol.

Mutant cyan fluorescent protein was prepared by transforming TOP10 competent cells (Invitrogen) with the vector of interest (ie, encoding the mutant fluorescent protein) according to the manufacturer's instructions. A pre-warmed volume of 1.5 L Luria-Bertani medium (LB) containing 100 μg / mL ampicillin was inoculated with 25 mL of a previously cultured overnight culture expressing the vector of interest. When the OD _{600 of the} cells reached a value of 0.6, protein production was induced by adding isopropyl-D-thiogalactopyranoside (IPTG, 1 mM) to the transformed cells. After culturing at 30 ° C. for 18 hours, the induced cells were collected by centrifugation and frozen. Cells were then suspended in 30 mL lysis buffer (50 mM Tris-HCl, 5 mM 2-mercaptoethanol, 1 mM PMSF and 0.02 mg / mL DNase) and sonicated. Finally, cell debris was removed by centrifugation (40,000 rpm, 1 h30 at 6 ° C.) to collect only the supernatant.

For purification of each mutant cyan fluorescent protein, the supernatant was filtered using a 0.22 μm filter, pH 7.5 phosphate buffer (30 mM NaH ₂ PO ₄ , 700 mM NaCl, 30 mM imidazole). Dilute to 2 times. This solution was then deposited for 1 hour in a column containing 5 mL of nickel nitroacetate (Ni-NTA) agarose (15 mL, Sigma). The column was then washed (30 mM NaH ₂ PO ₄ , 100 mM NaCl, 10 mM imidazole, pH 7.5) and the protein of interest was eluted (30 mM NaH ₂ PO ₄ , 100 mM NaCl, 150 mM imidazole, pH 7 .Five). Prior to storage at −20 ° C., each purified protein solution was subjected to a 2 mM concentration consisting of equimolar concentrations of CAPS, 2- (N-morpholino) ethanoic acid (MES) and bis-trispropane for spectroscopic experiments. And dialyzed against pH 7.4 buffer, then concentrated to obtain a protein solution with a concentration in the range of 150 μM.

  Next, the concentration of the purified protein was determined by a test using BSA (bovine serum albumin, 1 mg / mL) and bicinchoninic acid as standards.

  The purity of ECFPr was estimated to be better than 99% by mass spectrometry. The purity of mutants purified by the same protocol was estimated to be similar (no difference in behavior was observed in the electrophoresis gel).

II.3. Evaluation of fluorescence spectroscopy and pH sensitivity All spectroscopic measurements of fluorescence absorption and emission were performed on solutions of purified protein with concentrations not exceeding 10 μM. The protein concentration was generally 10 μM for both stationary spectroscopic measurements (absorption and emission spectra) and acquisition of fluorescence emission decay.

For pH studies, aliquots of concentrated solutions of the protein of interest were diluted in separate buffers that were pre-adjusted to the appropriate pH (measurement accuracy: 0.1 pH units). The nature of the buffer used depends on the pH range studied:
(i) For pH values above 5.5 (pH 5.5-11), the buffer used was MCBtP 33 mM (normal buffer) adjusted for pH by addition of H ₂ SO ₄ or NaOH (Aldrich). Equimolar mixture of MES, CAPS and bis-trispropane);
(ii) For pH values below 5.5 (pH 2.5-5.5), the buffer used is citric acid 50 mM / NaOH, also pH adjusted by addition of NaOH.

  Direct addition of concentrated acid to the fluorescent protein solution was avoided to avoid irreversible protein aggregation.

II.3.a. Stationary fluorescence spectroscopy UV-visible absorption spectra were performed on a Perkin Elmer Lambda 900 spectrometer using a quartz cuvette (Hellma) with a 1 cm thickness and black sidewalls.

  The fluorescence emission spectra of each purified protein was measured using a quartz cuvette (Hellma 105-251-QS, Hellma Ltd) with a thickness of 0.3 cm and a temperature controlled by a Fluorolog 3 HORIBA Jobin Yvon spectrum fluorometer (T = 20 ° C. ± 0.1 ° C.). The slit width of the excitation and emission monochromator was set to 1 nm. The spectra were then collected with an integration time of 1 second and 1 nm increments. The background noise emitted by the buffer was subtracted.

For each purified fluorescent protein, the intensity at 474 nm (the maximum value of the ECFPr emission spectrum) was measured from the fluorescence emission spectrum determined as a function of pH and its generation. These fluorescence intensities are then corrected for absorbance variation at 420 nm (excitation wavelength), which allows measurement of variation in quantum yield of fluorescence emission at 474 nm. The half-transition pH, pH _1/2, was then measured as the pH value at which the sum of these fluorescence intensities at pH 7.4 and pH 2.5, corresponding to the weakest fluorescence intensity, was halved.

II.3.b.Time-resolved fluorescence spectroscopy Fluorescence emission decay is achieved in a locked mode (MIRA 900) optically pumped by a 532 nm (10 W Verdi, Coherent) laser diode using a Ti: sapphire tunable laser as the excitation source Coherent, Watford, UK) using time correlated single photon counting (TCSPC).

The laser repetition rate pulse was reduced from 76 MHz to 3.8 MHz through a pulse picker (crystalline SiO2, APE, Berlin, Germany). After the pulse picker, an excitation wavelength of 420 nm was obtained by doubling the frequency of 840 nm laser radiation using a BBO crystal doubler. After doubling the frequency, laser excitation light was sent to the sample placed in a temperature-controlled multi-position sample holder. The average laser power at the sample was typically 1-1.5 μW (about 1-2 mm beam waist). Fluorescence decay curves were collected by a rapid electronic device (Ortec, Phillips & Tennelec). The instrument response function (IRF) obtained by measuring the light scattered by the LUDOX solution (Dupont) typically has a full width at half maximum of 60-70 ps. The excitation was vertically polarized and the sample fluorescence was passed through a polarizer directed at the magic angle (54.7 °) in front of the emission monochromator. In most experiments, this resolution is 6 nm, except for pH studies performed with ECFPr (H148D) where the monochromator spectral resolution was 24 nm. To obtain sufficient statistics, sample fluorescence and IRF were collected alternately over several tens of cycles: a total count of approximately 20.10. ⁶ was collected at a rate of about 10 ⁴ ct per second for each decay curve.

III. Development of biosensors with reduced pH sensitivity
III.1. Construction of pHAKAR biosensors pHAKAR biosensors with reduced pH sensitivity are Citrine and cyan fluorescent proteins (particularly including mutations 26R, 164H and 206K) as their energy acceptors and donors, and The AKAR 2.2 biosensor (Dunn et al., 2006), consisting of a biosensitive sensor sandwiched between these two proteins, itself consisting of a binding domain to phosphorylated amino acid (FHA) and a protein kinase A substrate sequence (called PKA) Built from. Phosphorylation of threonine by PKA at the substrate sequence leads to recognition of the latter by the FHA binding domain; this leads to a change in the biosensor conformation, which leads to an increase in the FRET signal between cyan fluorescent protein and Citrine .

  The nucleotide sequence encoding the AKAR2.2 biosensor was first inserted into the pcDNA3 vector (Life Technologies).

  Point mutations with the "QuikChange" kit (Stratagene) using primer couples of sequences SEQ ID NO: 49 and SEQ ID NO: 50 and couples of SEQ ID NO: 37 and SEQ ID NO: 38, respectively, followed by 65S and 148G mutations by pcDNA3-AKAR2 .2 introduced into the plasmid. Thus, the cyan fluorescent protein of the biosensor contains the 65S and 148G mutations of the present invention.

  Next, TagRFP monomer protein with orange fluorescent emission (Merzlyak et al., 2007) was cloned in place of Citrine as follows: Nucleotide sequences encoding biosensor mutant cyan fluorescent protein and biosensitive sensor , First cloned between the HindIII and SacI restriction sites into the pPROEXHTa vector (GibcoBRL); then the nucleotide sequence for the TagRFP protein was tagged with SEQ ID NO: 77 (5'ATTAGAGCTCATGGTGTCTAAGGGCGAA3 ') 5′ATAATGAATTCTTAATTAAGTTTGTGCCCC3 ′) sequence TagRFPr primer was used to amplify by PCR from a commercially available pTagRFP-C vector (Evrogen) and cloned between the SacI and EcoRI sites of the pPROEXHTa vector [ECFPr− of sequence SEQ ID NO: 82 Biosensitive sensor].

  The resulting protein kinase A biosensor, named pHAKAR, containing the fluorescent protein pair ECFPr / TagRFP of sequence SEQ ID NO: 82, and a biosensitive sensor, is then placed in the HindIII and EcoRI restriction sites of the pCDNA3 plasmid vector. Introduced. Each cloning was verified by sequencing.

  Plasmids encoding ECFPr (65S, 148G) protein (pECFPN1, Clontech) and a plasmid encoding pHAKAR biosensor (pcDNA3-pHAKAR) were amplified in E. coli and stored at a concentration of 1 μg / μL.

III.2. Fluorescence spectroscopy and evaluation of pH sensitivity of pHAKAR biosensor
BHK hamster cells were grown in 25 cm ² flasks in the presence of DMEM medium supplemented with 10% fetal calf serum (Gibco). During re-picking, cells were deposited on 25 mm diameter glass coverslips placed on the bottom of a 6-well plate and then 90% confluent lipotransfection with pECFPN1 or pcDNA3-pHAKAR plasmid according to manufacturer's protocol (4 μg plasmid and 2 μL Lipofectamine 2000; Life Technologies per 2 mL medium). Twenty-four hours after transfection, coverslips were mounted in attofluor metal chambers (Life Technologies). The medium used for the measurement contained 140 mM KCL, 15 mM MES buffer and 15 mM Hepes buffer, and its pH was adjusted to the desired value (7.4 or 5.9). The intracellular pH was adjusted to the external pH with ionophore and nigericin (final concentration 10 μM). After incubation with nigericin at 37 ° C. for 5 minutes, the transfected cells were placed on a microscope plate at 20 ° C.

Lifetime of fluorescence emitted by ECFPr protein containing at least 65S and 148G mutations incorporated into pHAKAR biosensors alone or in water, allowing identification of immersion objectives (x60, NA 1,2), mercury lamps and fluorescent cells Using a NIKON TE2000 microscope equipped with a fluorescence detection system (filters: Omega XF114-2 for ECFPr and TRITC-B-NTE Semrock for TagRFP), and excitation and detection means for fluorescence lifetime measurements Next observed at different pH values (7.40 and 5.9). Excitation was performed by a pulsed diode at 442 nm (LDH 440 and PDL 800D driver, PicoQuant) injected into a fibrous, C1 headscan type (Nikon) originally used for confocal microscopy. Headscan is guided by EZ-C1 software and allows spatial control of sample excitation. Under the objective lens, the fluorescence photons are filtered (Omega 480AF30 filter and two 458nm RazorEdge Long-Pass Semrock), followed by a retractable dichroic mirror (45 ° SWP-500, Lambda Research) in a microchannel plate (Hamamatsu). It was reflected toward. The signal was then collected by a photon counting module PicoHarp300 (Picoquant) that was also synchronized with the excitation laser pulse. Data was analyzed by SymPhoTime (Picoquant) software. Each cell was identified as a region of interest, and a fluorescence lifetime histogram was calculated from all pixels in this region. The total count was included between 1 and 5.10 ⁶ and the average life was calculated by SymPhoTime. For each pH condition, 5-10 cells were measured.

IV. Study of the effect of 65S mutation on quantum yield, fluorescence decay and photostability of ECFP cyan fluorescent protein According to the protocol described in Point II above, ECFPr, ECFPr (65S), ECFPr (72A, 145A and 148D ) And ECFPr (65S, 72A, 145A and 148D) proteins were produced, produced and purified.

IV.1. Analysis of spectroscopic data
Each fluorescence emission decay F (t) with corresponding instrument function (IRF) was analyzed individually using the maximum entropy method described by Merola et al. (1989) and Couprie et al. (1994). This analysis assumes that the experimental decay F (t) is the product of the following convolution:

Where g (t) is the measured IRF and I _m (t) represents the fluorescence emission law I (t) continuous to infinitely short excitation. The analysis assumes that the fluorescence decay law consists of a number of exponential terms. Therefore, the total attenuation corresponds to the following formula:

Where α (t) is the normalized pre-exponential amplitude (i.e.

) And I ₀ is an indeterminate factor that incorporates the experimental conditions of measurement. The time shift between F (t) and g (t) is also optimized, and reduced χ ² values are found in the range 0.97 to 1.05, with residuals and autocorrelation functions distributed randomly.

From the distribution of fluorescence lifetimes α (t) recovered by this method, the integration of each individual peak observed in the distribution results in a small number of individual components (τ _i ) and their corresponding pre-exponential amplitudes (c _i )was gotten. The distribution α (t) is the mean fluorescence lifetime <τ _f >, which should be proportional to the fluorescence quantum yield (Value B., 2006):

Was used to calculate

The measurement uncertainties for c _i , τ _i and <τ _f > were determined from standard deviations from several repeated identical experiments.

IV.2. Synchrotron radiation circular dichroism was measured using a 11 μm calcium fluoride circular cuvette (Wien et al., 2005) (Hellma) and using Solil synchrotron (Gif sur Yvette, France) (Giuliani et al., 2009). Performed at DISCO beamline.

Typically, the protein concentration was 8 mg / L at pH 2.5 and 18 mg / L at pH 7.4. All samples were prepared the day before with their buffer (30 mM CAPS pH 7.4, 30 mM MES and 30 mM bis-trispropane and pH 2.5 30 mM citrate). For each protein, three scans from 170 to 280 nm at 1 nm intervals per second were recorded and then averaged. Three consecutive scans of baseline (using buffer) were similarly obtained and averaged. Experiments were recorded at 25 ° C. for all proteins. The buffer spectrum was subtracted from that of the corresponding sample. The 260-270 nm region was set to zero and the resulting spectrum was calibrated with CSA (D-10-camphosulfonic acid) using CDtool software (Lees et al., 2004). The average circular dichroism per residue is expressed as M ⁻¹ .cm ⁻¹ (Kelly et al., 2005). Secondary structure determination was performed by DICHROWEB (Whitmore et al., 2004) using the CDSSTR and CONTINLL algorithms and the SP175 database (Lees et al., 2006). The two algorithms provide similar results. Results were obtained using the CDSSTR algorithm. NRMSD adjustment parameters are in the range of 0.030 to 0.050 for all proteins.

IV.3. Photobleaching experiment
IV.3.a. Labeling of agarose beads with cyan fluorescent protein Nickel-loaded agarose beads (Sigma) were labeled with the mutant recombinant protein described above. 100 μL of the deposited beads were prewashed, equilibrated with phosphate buffer (pH 7.5), and incubated with 1-5 μM of purified protein in a total volume of 1 mL for 1 hour with gentle agitation in the cold. The beads were then centrifuged at 5000 rpm for 5 minutes, washed twice and resuspended in PBS. For photobleaching, several microliters of bead suspension was deposited on a microscope cover glass with a diameter of 25 mm.

IV.3.b. Expression of cytosolic cyan fluorescent protein MDCK cells grown on 25 mm diameter coverslips using Lipofectamine 2000 according to manufacturer's recommendations (Invitrogen) encode the cyan fluorescent protein of interest Transfections were transiently transfected with eukaryotic expression plasmids. These cells were studied 24-48 hours after their transfection.

IV.3.c.Irradiation and imaging conditions Using an epifluorescence microscope equipped with a water immersion objective (× 60, NA 1.2; Nikon), HBO 100 W Hg lamp, and ECFP dichroic filter for detecting fluorescence ( Using Omega XF114-2), a fluorescent photobleaching experiment of cyan fluorescent protein was performed at 20 ° C. ± 0.5 ° C. The irradiation power was adjusted with a neutral density filter, the maximum power measured on the sample without any attenuation was approximately 200 μW (FieldMaster 13M41 detector, Coherent) and the irradiation field radius was estimated to be 185 μm, above the sample. Resulted in an average illuminance of about 0.2 W / cm2. Single beads or groups of MDCK cells were placed in the center of the imaging field, fluorescence intensity was measured with a cooled CCD camera (ORCA-AG Hamamatsu), and quantified using NIH ImageJ software. No significant dependence of the photobleaching rate measured as a function of bead size or bead label density could be observed.

IV.3.d. Modeling and analysis of photobleaching experiments Kinetic model The cyan fluorescent protein ECFP is a photoactivated reversible between two states characterized by rate constants k _off and k _on , a fluorescent state and a non-fluorescent state It is postulated that it undergoes a general reaction.

  This model ignores thermal relaxation between these two states, as well as irreversible photobleaching (depending on the experimental conditions tested here, they occur on a slower time scale), and the two forms of identical Assume absorption. This results in the following formula:

x, set the relative fraction of fluorescence

Taking x ₀ = 1 (when the system is in thermal equilibrium in the dark, there is no non-fluorescent state at time 0):

That is, as a result of rapid irradiation, the molar fraction of the fluorescent state is time constant τ _Rev = 1 / (k _on + k toward the steady level y ₀ = k _on / (k _on + k _off ) _off ) decreases monoexponentially.

Adjustment of reversible photobleaching experiment After initial intensity standardization to 1, model transient photobleaching curve

Where y ₀ represents the steady-state fluorescence level and y ₁ represents the irreversible photobleaching rate constant assuming a linear law (y ₁ values are generally ≈-1 * 10 ^-3 s ^-1 Which corresponds to the resulting irreversible photobleaching rate constant (see FIG. 22), y ₂ and τ _Rev are the relative amplitude and time constant of reversible fading, respectively.

Fluorescence recovery after transient photobleaching is accelerated under irradiation.
After normalizing the initial intensity to zero and normalizing the maximum intensity to 1, the fluorescence intensity recovery curve was adjusted with the same analytical model:

The relaxation time τ _Back for fluorescence recovery shows excellent agreement with the relaxation time τ _Rev for fluorescence loss (FIG. 28).

All rate constants k _on and k _off depend on the irradiation intensity The rate constants k _on and k _off are obtained directly from y ₀ and τ _Rev :

The k _on and k _off 2 two states kinetic model assuming linear dependence of the irradiation power, the relaxation time tau _Rev should depend linearly to excitation power (or, irradiation expressed in W / cm @ 2), This should not be the case for the reversible fluorescence-depleted fraction (1-y ₀ ) (this has been almost verified for low excitation powers; see especially FIG. 28).

Quantum yield of cyan fluorescent protein photoconversion The quantum yields φ _off and φ _on of the photoconversion reaction of cyan fluorescent protein ECFPr are given by the following equations:

_Where k _exc is the excitation rate per molecule and is given by the product of the effective absorption section σ _exc × photon flux N _exc at the excitation wavelength λ _exc : k _exc = σ _exc (cm ² ) × N _exc (Photon / second / cm ² )

For ECFPr cyan fluorescent protein, assuming I _exc = 0.05 W / cm2 and λ _exc = 440 nm and assuming ε _on = ε _off ≈30000 M ^-1 cm ^-1 , k _exc = 13s ^-1 is obtained, k _off = 0.18 s ⁻¹ and k _on = 0.77 s ⁻¹ , giving φ _off = 1.4% and φ _on = 6.1%.

B-Result
I. Comparison of pH sensitivity of ECPF as a function of pH and mutant cyan fluorescent protein according to the invention For the purpose of producing cyan fluorescent proteins with reduced pH sensitivity, especially to acidic pH, we We wanted to study the effect of various mutations on the pH sensitivity of ECFP.

  Therefore, a variety of single mutations were introduced into ECFP in recombinant form (ECFPr) to reduce the fluorescence intensity loss and fluorescence lifetime normally observed with this protein when the pH goes from basic to acidic pH. Their effects on reduction were studied. The results of this study are listed in Table 3 below.

  In fact, as seen in Figure 1 and Table 3 (below), ECFPr shows a 50% loss of fluorescence intensity and a 33% decrease in fluorescence lifetime when the pH drops from 7.4 to 5.5. . Spectral alterations are also seen when the pH becomes acidic. These properties strongly limit the reliability of the use of ECFPr in quantitative imaging methods in living cells such as FRET because they are particularly sensitive to slight variations in fluorescence signal Because. Indeed, the intracellular pH varies depending on the cellular compartment in which ECFPr is solubilized and on the experimental conditions being tested.

Furthermore, the ECFPr half-transition pH (pH _1/2 ) measured by the inventors is 5.6, and its fluorescence lifetime at neutral pH is 2.5 ns. It should be noted that the pH _1/2 value of ECFPr determined by the inventors is different from the value which is approximately 4.6-4.7 as described in the literature. Only an objective analysis as reported herein makes it possible to adequately study the true difference in pH _1/2 between the proteins of the invention and those of the prior art.

  Therefore, in order to solve the pH sensitivity problem of ECFP, we first introduced a single mutation at position 148 of ECFPr.

  The introduction of the 148D mutation produced a cyan fluorescent protein that showed an approximately 50% loss in fluorescence intensity and a 40% decrease in fluorescence lifetime when the pH was changed from 7.4 to 5.5. Therefore, its fluorescence lifetime at neutral pH reaches 3.3 ns, whereas that of ECFPr is 2.5 ns, which is higher than that of ECFPr (Table 3). Thus, although this mutation did not reduce fluorescence intensity and lifetime loss at acidic pH compared to ECFPr, it significantly improves lifetime at neutral pH.

Next, we introduced the 148G mutation into ECFPr, thereby producing ECFPr (148G) protein. This protein shows only a 18% loss of fluorescence intensity and a 6% decrease in fluorescence lifetime at acidic pH. Its pH _1/2 is 4.9 and its fluorescence lifetime at neutral pH is 3.37 ns (FIG. 2 and Table 3). Thus, the 148G mutation allows a reduction in the pH sensitivity of ECFP.

  We next tested other mutations at position 148 of ECFP, particularly mutations 148E, 148S and 148A.

ECFPr (148E) protein exhibits a 27% loss in fluorescence intensity and an 18% decrease in fluorescence lifetime between pH 7.4 and pH 5.5. Its pH _1/2 is 5.1 and its fluorescence lifetime at neutral pH is 3.15 ns (Figure 15 and Table 3).

The ECFPr (148S) protein thus generated shows only a 9% loss of fluorescence intensity and a 15% decrease in fluorescence lifetime between pH 7.4 and pH 5.5. In addition, the pH _1/2 of this protein is 4.5 and its fluorescence lifetime at neutral pH is 3.17 ns (Figure 3 and Table 3). Thus, the introduction of the 148S mutation into ECFP allows the acquisition of cyan fluorescent protein with reduced pH sensitivity.

ECFPr (148A) protein shows only a 7% loss of fluorescence intensity and a 4% decrease in fluorescence lifetime between pH 7.4 and pH 5.5. Furthermore, the pH _1/2 of this protein is 4.5, and its fluorescence lifetime at neutral pH is 3.15 ns (Figure 4 and Table 3). Thus, the introduction of the 148A mutation into ECFP allows the acquisition of cyan fluorescent protein with reduced pH sensitivity.

  Thus, the amino acid at position 148 of ECFP appears to play an important role in the pH sensitivity of this protein.

Subsequently, the inventors focused on the amino acid at position 65 of ECFP. They introduced the 65S mutation by directed mutagenesis, thereby producing ECFPr (65S) protein. When the pH goes from 7.4 to 5.5, this protein exhibits advantageous properties because its loss of fluorescence intensity is only 15% and the decrease in fluorescence lifetime is 16%. The pH _1/2 of this protein is 4.5 and its fluorescence lifetime is 3.3 (Figure 5 and Table 3). Thus, the amino acid at position 65 also appears to affect the pH sensitive properties of ECFP.

  In addition to those introduced at positions 148 or 65, other mutations were also evaluated. These were selected from among mutations 72A, 145A and 206K.

Thus, ECFPr (72A, 145A, 148D) cyan fluorescent protein exhibits spectral modifications like ECFPr. When the pH goes from 7.4 to 5.5, its loss in fluorescence intensity and lifetime is 33% and 32%, respectively, and therefore less than that of ECFPr. Its half-transition pH (pH _1/2 ) is 5.2 (FIG. 7 and Table 3). Nevertheless, the reduction in pH sensitivity conferred by a combination of these mutations is less advantageous than that conferred by a single mutation 148G, 148S, 148A or 65S.

Therefore, we exchanged mutation 148D for mutation 148G, thereby generating ECFPr (72A, 145A, 148G). This combination of mutations allows a reduction in pH sensitivity, as the fluorescence intensity and lifetime depletion are only 13% and 3% when the pH goes from 7.4 to 5.5, respectively. Furthermore, the pH _1/2 of this protein is less than that of ECFPr and its fluorescence lifetime at neutral pH is increased (tau = 3.13 ns) (FIG. 8 and Table 3).

  The combination of the 65S mutation and the 72A and 206K mutations also allows for a reduction in pH sensitivity compared to ECFP (see Figure 12 and Table 3). However, the loss of fluorescence intensity and lifetime observed with this protein (ECFPr (65S, 72A, 206K)) exceeds that observed with the single mutation 65S.

  We therefore tested the double mutations at positions 148 and 65 in the presence or absence of additional mutations, in this case mutations 72A and 206K.

  The introduction of these double mutations in the presence or absence of additional mutations allows a significant reduction in the pH sensitivity of ECFP and even eliminates it (see Table 3).

  In fact, when the pH goes from basic pH to acidic pH, the proteins ECFPr (65S, 72A, 145A, 148D), ECFPr (65S, 72A, 148D, 206K) and ECFPr (65S, 72A, 148G, 206K), ECFPr The fluorescence intensity and lifetime loss of (65S, 148G), ECFPr (65S, 148D) and ECFPr (65S, 148S) is almost 0%, if not completely zero.

Among these proteins, ECFPr showing only double mutations (65S, 148G) and (65S, 148S), as well as ECFPr (65S, 72A, 148G, 206K) are all produced by the inventors. It exhibits the best properties among mutant cyan fluorescent proteins: in fact, the observed fluorescence intensity and lifetime loss is zero when the pH goes from 7.4 to 5.5, and their respective pH _1/2 is 3.4; 3.6 and 3.1 and their respective average fluorescence lifetimes at neutral pH are 4.12; 3.86 and 4.06 ns.

  Furthermore, with respect to fluorescence lifetime, ECFPr (65S, 148G) is the best among all the cyan fluorescent proteins produced by the inventors.

  In conclusion, the introduction of a single mutation at position 148 and / or 65 in ECFP, more specifically the mutation at 148G / A and / or 65S, is a cyan fluorescent protein whose pH sensitivity is reduced if not eliminated. Enables the generation of The use of such proteins in quantitative imaging applications allows for more reliable quantification of the fluorescent signal that they emit.

II. Study of pH sensitivity of new biosensors At physiological pH (7.4), the average lifetime of fluorescence emitted by the cyan fluorescent protein ECFPr, which is incorporated into the pHAKAR biosensor and contains the 65S and 148G mutations among others, is We found that there was a 16% reduction compared to ECFPr (65S, 148G) protein expressed alone (3.32 ns vs 3.96 ns). This decrease in fluorescence lifetime is not due to the presence of the additional mutations 26R, 164H and 206K, which are silent mutations in terms of the photophysical properties of the cyan fluorescent protein, but the energy between the cyan fluorescent protein and TagRFP. Associated with metastasis.

  Nevertheless, when the intracellular pH is reduced to 5.9, the average lifetime of fluorescence of the pHAKAR biosensor mutant protein ECFPr is only slightly changed from 3.32 ns to 3.23 ns, and the pHAKAR biosensor Emphasizes the lack of pH sensitivity.

  Thus, considering these results, a pair of fluorescent proteins ECFPr (65S, 148G) that can further include additional mutations and TagRFP that do not affect its photophysical properties have reduced pH sensitivity. It seems to constitute a FRET pair that demonstrates its performance.

III. Study of the effect of 65S mutation on quantum yield, fluorescence decay and photosensitivity of cyan fluorescent protein As mentioned above, introduction of 65S mutation has the effect of reducing the pH sensitivity of ECFP cyan fluorescent protein . The inventors have also discovered that the introduction of this mutation further increases the quantum yield, simplifies the kinetics of fluorescence, and improves the photostability of this protein against photobleaching.

III.1. 65S mutation improves the homogeneity and quantum yield of cyan fluorescent protein. The fluorescent absorption and emission properties of purified proteins ECFPr and ECFPr (72A, 145A, 148D) are reduced to 65S at neutral pH and room temperature. It was compared with those of the proteins containing the mutations, namely the proteins ECFPr-65S and ECFPr (65S, 72A, 145A, 148D). The absorption and fluorescence spectra of these cyan proteins are actually very similar, showing the bimodal absorption and emission spectra typical of indole-type chromophores, with two absorption maxima at 430nm and 445nm and at 474nm and 500nm. The present inventors have found that these have two emission maximum values (FIG. 21). Considering experimental uncertainties, very close absorption coefficients were found for all proteins studied in the range of 32000 ± 4000 M ⁻¹ .cm ⁻¹ at 430 nm (table in FIG. 22). Despite close spectral similarities, these proteins differ significantly in their quantum yield (Figure 22) and in their fluorescence lifetime distributions as calculated from their fluorescence decay (Figure 23). . These lifetime distributions include, in all cases, a major, long fluorescence lifetime associated with a variable amount of short components (FIG. 22). The ECFPr (72A, 145A, 148D) protein has an enhanced quantum yield compared to that of the ECFPr protein, but retains non-uniform fluorescence and its longest lifetime is only 64% of the amplitude of total amplitude decay (FIGS. 22 and 23).

  The 65S single mutation introduced by the method of the present invention significantly improves the performance of ECFPr and ECFPr (72A, 145A, 148D) proteins. The fluorescence quantum yield of the protein of ECFPr-65S is increased by 48% compared to ECFPr, and that of ECFPr (65S, 72A, 145A, 148D) is increased by 25% compared to ECFPr (72A, 145A, 148D) ( (Figure 22). The 65S mutation also results in considerable simplification of the fluorescence lifetime distribution (Figure 23). For the ECFPr (72A, 145A, 148D) protein, the 65S mutation leads to high fluorescence quantum yield and average lifetime, and the fluorescence decay follows near monoexponential dynamics with 83% lifetime amplitude (FIG. 22).

III.2. 65S mutation suppresses the intermediate form of cyan fluorescent protein detected during acid transfer Detailed analysis of the fluorescence absorption and emission spectra of ECFPr and its mutants during acid transfer Provide a large amount of evidence to support the existence of intermediate forms. Specifically, in the case of ECFPr, a shift to red at the maximum value of the fluorescence emission spectrum is observed following acidification of the medium to pH 4.7 (FIG. 24). As the pH further decreases, the emission maximum shifts again to blue. This behavior cannot be explained by a simple transition between the two states, and the presence of an intermediate state clearly distinct from the two neutral and denatured states at a highly acidic pH whose fluorescence emission shifted to red Providing evidence of This “red” intermediate disappears when the 65S mutation is introduced into ECFPr (FIG. 25C). Similarly, for the protein ECFPr (72A, 145A, 148D), there is an intermediate below pK _1/2 that is absorbed in blue and emits light (Figure 25B and Figure 26B); this isomerized intermediate The condition (more known by the name of the cis-trans isomer) becomes undetectable after the introduction of the 65S mutation (FIGS. 25D and 26D). In fact, the fluorescence absorption and emission spectra of ECFPr (65S, 72A, 145A, 148D) proteins show a mixture of both neutral and acidic forms without any intermediate spectral disruption. The loss of fluorescence intensity and the fine structure of the absorption and emission spectra, as well as the spectral shift to blue, occur virtually simultaneously in a very narrow pH range.

III.3. 65S mutation reduces the rate and amplitude of reversible photobleaching of cyan fluorescent proteins Many natural or genetically modified fluorescent proteins are induced by light between two optically distinct states Undergoes reversible conversion (also called reversible photoswitching protein or RSFP). It is further appreciated that such reversible photoreactions are common to the majority of fluorescent proteins to varying degrees, and this can have great consequences for their use in biological imaging: In standard FRET applications, the fluorophore should eliminate such photoreactions as much as possible. Nevertheless, quantitative data regarding the reversible light conversion properties of cyan and yellow fluorescent proteins is rather rare. These reactions are generally easier to observe with purified and immobilized fluorescent proteins in order to eliminate interference due to Brownian diffusion or movement of living cells expressing these proteins. is there.

  The present inventors were able to observe that the purified protein ECFPr that binds to agarose beads undergoes a remarkably reversible photobleaching that is transient under irradiation in its chromophore absorption band. Thus, under abrupt wide-field illumination using the maximum power of a mercury lamp, the fluorescence intensity decreases by 23% after a single exponential time constant of less than 1 second (FIGS. 22 and 27). After this transient response, there is a slower decrease in fluorescence intensity of about 0.1% per second, possibly due to irreversible photobleaching (see below). If the irradiation time is kept short enough, and after a few minutes in complete darkness, the fluorescence intensity returns to its initial level (FIG. 27A).

We further observed that the recovery of ECFPr fluorescence after transient fading was accelerated by moderate illumination at the same wavelength (FIG. 28). This indicates that the return of ECFPr to its fluorescent state is also activated by light. Thus, the steady state level of fluorescence achieved after a few seconds of irradiation must correspond to a steady state regime, where both the “off” (non-fluorescent state) and “on” (fluorescent state) photoreactions are equal. Happens at speed. To describe this system, a kinetic model containing two minimum states can be used: this model has two rate constants k _off and k _on describing the elementary reactions of reversible fading and photoactivation reversion, respectively. It is characterized by. According to this model, the apparent relaxation time τ _rev in the fading experiment is the reciprocal of the sum of the two rate constants (k _on + k _off ) ⁻¹ , but the steady-state level of fluorescence intensity reached after a few seconds is k _on / (k _on + k _off ), from which the rate constants k _on and k _off can be obtained (Table 4). From these two rate constants, the photoconversion quantum yield in both directions can also be estimated: the obtained values φ _off = 1% and φ _on = 6% are reversible photoswitching proteins that are highly effective in ECFPr (RSFP).

Transient fading of other cyan fluorescent proteins with or without the 65S mutation was also studied according to the same experimental protocol. As a result of abrupt irradiation, all fluorescent proteins undergo a reversible decrease in fluorescence intensity with a similar time scale of a few seconds but with very different amplitudes (FIG. 27). Thus, it is noted that the ECFPr (72A, 145A, 148D) protein shows a significant decrease of 33%. Even more notably, the 65S mutation introduced in ECFPr or ECFPr (72A, 145A, 148D) clearly reduces the amplitude of the transient decrease to less than 3% in each case (FIG. 27B). Comparing the rate constants k _on and k _off of each of these proteins (Table 4), the protein containing the 65S mutation is 10 times lower than that of the protein lacking this mutation. _It is noted that there is only a moderate change in photoactivation recovery (Table 4). Thus, the principle action of the 65S mutation is to significantly slow down the basic rate k _off of reversible fading, which leads to a reduction in the amplitude of transient fading.

  Photoreactions of cyan fluorescent proteins can also be observed in MDCK cells that express these proteins in their cytosol (FIG. 29). However, using the same wide-field illumination conditions, the amplitude of response appears to be significantly reduced compared to that obtained in vitro, and the relaxation time is much shorter (Table 4), This may arise from the conditions of acquisition. Nevertheless, comparison of different cyan fluorescent proteins reveals a trend that is very similar to that observed in vitro, ie, the 65S mutation strongly reduces the transient response (Figure 29).

III.4. 65S mutation slows irreversible photobleaching of cyan fluorescent protein
The irreversible fading reaction of cyan fluorescent protein with or without the 65S mutation was also studied. To do so, the same but long-term wide-field irradiation conditions were used for both immobilized agarose beads and cell cytosol. All cyan fluorescent proteins studied show irreversible fluorescence loss of 85% to 95% after 30 minutes irradiation at maximum power under a mercury lamp. The fluorescence intensity decay is almost exponential (FIG. 30) with less than 1% intensity recovery in the dark and no detectable photoactivation recovery until 10 minutes after irradiation cessation. Experiments performed with immobilized agarose beads and cells gave fairly similar irreversible fading time constants (Figure 22): therefore, in all cases, the single mutation 65S was associated with the proteins ECFPr and ECFPr (72A, 145A, 148D) significantly slows irreversible fading (Table 1). The ECFPr (72A, 145A, 148D) protein undergoes slightly faster fading than ECFPr, thereby demonstrating that the protein has reduced photostability for both reversible and irreversible reactions.

  Thus, the effect of the 65S mutation is to improve the light stability of the cyan fluorescent protein in response to irradiation.

Conclusion Therefore, we were able to observe a considerable effect of the 65S mutation on the photophysical properties of cyan fluorescent protein. This specific mutation, in addition to reducing the pH sensitivity of these proteins, increases their quantum yield, reduces their fluorescent kinetic complexity, and inhibits reversible photoreactions and slows irreversible photobleaching. Enable.

Thus, more specifically, cyan fluorescent proteins containing the 65S mutation allow for a more precise and more sensitive quantitative analysis of the fluorescent signal and thus a more reliable analysis, so that in live cells It can be used in imaging studies by Forster resonance energy transfer (FRET) or fluorescence lifetime imaging microscopy (FLIM).
(Reference)

Claims (17)

A step wherein a single or two mutations are introduced into a protein sequence comprising the sequence of SEQ ID NO: 2, said mutation being introduced at position 148 and / or 65 of the sequence of SEQ ID NO: 2, and- The amino acid at position 65 is substituted with serine and / or the amino acid at position 148 is substituted with glycine, alanine or serine, step a)
A method for producing a cyan fluorescent protein exhibiting reduced pH sensitivity. Another mutation selected from the group of 9G, 11I, 19E, 26R, 68L, 87V, 164H, 167A, 172T, 175G, 194I and 206K and combinations thereof are introduced into the sequence of SEQ ID NO: 2) b)
The method of claim 1, further comprising: step b) is performed before or after step a). The cyan fluorescent protein obtained by the method according to claim 1 or 2 , characterized in that the sequence of the cyan fluorescent protein comprises at least the 65S and 148G mutations .   A nucleic acid encoding the cyan fluorescent protein according to claim 3.   A recombinant vector comprising the nucleic acid according to claim 4.   A host cell expressing the cyan fluorescent protein according to claim 3.   A biosensor comprising the cyan fluorescent protein according to claim 3. 8. The biosensor according to claim 7, wherein the biosensor includes a fluorescent protein whose absorption spectrum partially overlaps with the emission spectrum of the protein according to claim 3. The fluorescent protein whose absorption spectrum partially overlaps with the emission spectrum of the protein according to claim 3, the fluorescent protein YFP, Topaz, EYFP, YPET, SYFP2, Citrine, Venus, cp-Venus, Kusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato, dTomato-Tandem, DsRed, DsRed2, DsRed-Express (T1), DsRed-Express2, DsRed-Max, DsRed-Monomer, TagRFP, TagRFP-T, mRuby, mApple, mStrawberry, AsRed2, Bio according to claim 8 , characterized in that it is selected from mRFP1, JRed, mCherry, eqFP611, tdRFP611, HcRed1, mRaspberry, tdRFP639, mKate, mKate2, Katushka, tdKatushka, HcRed-Tandem, mPlum and AQ143. sensor. 10. The biosensor according to claim 9 , wherein the fluorescent protein whose absorption spectrum partially overlaps with the emission spectrum of the protein according to claim 3 is TagRFP. The cyan fluorescent protein according to claim 3, characterized in that directly connected to the biosensor, biosensor according to claim 7. The cyan fluorescent protein according to claim 3 or the fluorescent protein whose absorption spectrum partially overlaps with the emission spectrum of the protein according to claim 3 is directly linked to a biosensor. The biosensor according to any one of claims 8 to 10 , characterized in that The cyan fluorescent protein according to claim 3, characterized in that the indirectly coupled to the biosensor, biosensor according to claim 7. The cyan fluorescent protein according to claim 3 or the fluorescent protein whose absorption spectrum partially overlaps with the emission spectrum of the protein according to claim 3 is indirectly linked to a biosensor. The biosensor according to any one of claims 8 to 10, characterized in that Fluorescence correlation spectroscopy (FCS) and its variants, Forster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), fluorescence lifetime imaging microscopy (FLIM), fluorescence redistribution after photobleaching (FRAP), light Fluorescence loss induced by fading (FLIP), time correlated single photon counting (TCSPC), anisotropy and fluorescence bleaching, photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy ( STORM), a cyan fluorescent protein according to claim 3, a nucleic acid according to claim 4, and a nucleic acid according to claim 5 in a quantitative imaging application selected from Stimulated Emission Suppression Microscopy (STED) Use of a recombinant vector, a host cell according to claim 6, and a biosensor according to any one of claims 7 to 14 . A cyan fluorescent protein according to claim 3, a nucleic acid according to claim 4, a recombinant vector according to claim 5, a host cell according to claim 6 in a method for screening compounds and / or cells. Use of a biosensor according to any one of claims 7 to 14 . In a toxicology, genotoxicity or environmental pollution detection test, the cyan fluorescent protein according to claim 3, the nucleic acid according to claim 4, the recombinant vector according to claim 5, the host cell according to claim 6, Use of the biosensor according to any one of claims 7 to 14 .